Orally active salts with tyrosine kinase activity

ABSTRACT

The present invention relates to orally active salts of compounds which inhibit, regulate and/or modulate tyrosine kinase signal transduction, compositions which contain these compounds, and methods of using them to treat tyrosine kinase-dependent diseases and conditions, such as angio-genesis, cancer, tumor growth, atherosclerosis, age related macular degeneration, diabetic retinopathy, inflammatory diseases, and the like in mammals.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to orally active salts of compoundswhich inhibit, regulate and/or modulate tyrosine kinase signaltransduction, compositions which contain these compounds, and methods ofusing them to treat tyrosine kinase-dependent diseases and conditions,such as angiogenesis, cancer, tumor growth, atherosclerosis, age relatedmacular degeneration, diabetic retinopathy, inflammatory diseases, andthe like in mammals.

[0002] Tyrosine kinases are a class of enzymes that catalyze thetransfer of the terminal phosphate of adenosine triphosphate to tyrosineresidues in protein substrates. Tyrosine kinases play critical roles insignal transduction for a number of cell functions via substratephosphorylation and have been shown to be important contributing factorsin cell proliferation, carcinogenesis and cell differentiation.

[0003] Tyrosine kinases can be categorized as receptor type ornon-receptor type. Receptor type tyrosine kinases have an extracellular,a transmembrane, and an intracellular portion, while non-receptor typetyrosine kinases are wholly intracellular.

[0004] Both receptor-type and non-receptor type tyrosine kinases areimplicated in cellular signaling pathways leading to numerous pathogenicconditions, including cancer, psoriasis and hyperimmune responses.

[0005] Several receptor-type tyrosine kinases, and the growth factorsthat bind thereto, play a role in angiogenesis, although some maypromote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol.129:895-898, 1995). One such receptor-type tyrosine kinase is fetalliver kinase 1 or FLK-1. The human analog of FLK-1 is the kinase insertdomain-containing receptor KDR, which is also known as vascularendothelial cell growth factor receptor 2 or VEGFR-2, since it bindsVEGF with high affinity. Finally, the murine version of this receptorhas also been called NYK (Oelrichs et al., Oncogene 8(1):11-15, 1993).VEGF and KDR are a ligand-receptor pair that play an important role inthe proliferation of vascular endothelial cells, and the formation andsprouting of blood vessels, termed vasculogenesis and angiogenesis,respectively.

[0006] Angiogenesis is characterized by excessive activity of vascularendothelial growth factor (VEGF). VEGF is actually comprised of a familyof ligands (Klagsburn and D'Amore, Cytokine & Growth Factor Reviews7:259-270, 1996). VEGF binds the high affinity membrane-spanningtyrosine kinase receptor KDR and the related fms-like tyrosine kinase-1,also known as Flt-1 or vascular endothelial cell growth factor receptor1 (VEGFR-1). Cell culture and gene knockout experiments indicate thateach receptor contributes to different aspects of angiogenesis. KDRmediates the mitogenic function of VEGF whereas Flt-1 appears tomodulate non-mitogenic functions such as those associated with cellularadhesion. Inhibiting KDR thus modulates the level of mitogenic VEGFactivity. In fact, tumor growth has been shown to be susceptible to theantiangiogenic effects of VEGF receptor antagonists. (Kim et al., Nature362, pp. 841-844, 1993).

[0007] Solid tumors can therefore be treated by tyrosine kinaseinhibitors since these tumors depend on angiogenesis for the formationof the blood vessels necessary to support their growth. These solidtumors include histiocytic lymphoma, cancers of the brain, genitourinarytract, lymphatic system, stomach, larynx and lung, including lungadenocarcinoma and small cell lung cancer. Additional examples includecancers in which overexpression or activation of Raf-activatingoncogenes (e.g., K-ras, erb-B) is observed. Such cancers includepancreatic and breast carcinoma. Accordingly, inhibitors of thesetyrosine kinases are useful for the prevention and treatment ofproliferative diseases dependent on these enzymes.

[0008] The angiogenic activity of VEGF is not limited to tumors. VEGFaccounts for most of the angiogenic activity produced in or near theretina in diabetic retinopathy. This vascular growth in the retina leadsto visual degeneration culminating in blindness. Ocular VEGF mRNA andprotein are elevated by conditions such as retinal vein occlusion inprimates and decreased pO₂ levels in mice that lead toneovascularization. Intraocular injections of anti-VEGF monoclonalantibodies or VEGF receptor immunofusions inhibit ocularneovascularization in both primate and rodent models. Regardless of thecause of induction of VEGF in human diabetic retinopathy, inhibition ofocular VEGF is useful in treating the disease.

[0009] Expression of VEGF is also significantly increased in hypoxicregions of animal and human tumors adjacent to areas of necrosis. VEGFis also upregulated by the expression of the oncogenes ras, raf, src andmutant p53 (all of which are relevant to targeting cancer). Monoclonalanti-VEGF antibodies inhibit the growth of human tumors in nude mice.Although these same tumor cells continue to express VEGF in culture, theantibodies do not diminish their mitotic rate. Thus tumor-derived VEGFdoes not function as an autocrine mitogenic factor. Therefore, VEGFcontributes to tumor growth in vivo by promoting angiogenesis throughits paracrine vascular endothelial cell chemotactic and mitogenicactivities. These monoclonal antibodies also inhibit the growth oftypically less well vascularized human colon cancers in athymic mice anddecrease the number of tumors arising from inoculated cells.

[0010] Viral expression of a VEGF-binding construct of Flk-1, Flt-1, themouse KDR receptor homologue, truncated to eliminate the cytoplasmictyrosine kinase domains but retaining a membrane anchor, virtuallyabolishes the growth of a transplantable glioblastoma in mice presumablyby the dominant negative mechanism of heterodimer formation withmembrane spanning endothelial cell VEGF receptors. Embryonic stem cells,which normally grow as solid tumors in nude mice, do not producedetectable tumors if both VEGF alleles are knocked out. Taken together,these data indicate the role of VEGF in the growth of solid tumors.Inhibition of KDR or Flt-1 is implicated in pathological angiogenesis,and these receptors are useful in the treatment of diseases in whichangiogenesis is part of the overall pathology, e.g., inflammation,diabetic retinal vascularization, as well as various forms of cancersince tumor growth is known to be dependent on angiogenesis. (Weidner etal., N. Engl. J. Med., 324, pp. 1-8, 1991).

[0011] Although quinolinyl-indole compounds have been previouslyreported to be useful as tyrosine kinase inhibitors (see WO 01/29025;published Apr. 26, 2001), a need still exists for forms of thesecompounds that can be readily administered to patients, especiallyorally active, soluble forms of these compounds. Accordingly, theidentification of orally active salts of compounds which specificallyinhibit, regulate and/or modulate the signal transduction of tyrosinekinases is desirable and is an object of this invention. The salts ofthe present invention have unexpectedly enhanced pharmokineticproperties as compared to compounds previously reported.

SUMMARY OF THE INVENTION

[0012] The present invention relates to salts of compounds that arecapable of inhibiting, modulating and/or regulating signal transductionof both receptor-type and non-receptor type tyrosine kinases. The saltsof the instant invention comprise salts of generic Formula I:

DESCRIPTION OF THE FIGURES

[0013]FIG. 1: X-ray powder diffraction pattern of the free base of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (1-10).

[0014]FIG. 2: X-ray powder diffraction pattern of crystalline forms ofthe mesylate salt of 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (A) Salt 1-10A(B) Salt 1-10B.

[0015]FIG. 3: X-ray powder diffraction pattern of crystalline HCl saltof3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one(1-11C).

DETAILED DESCRIPTION OF THE INVENTION

[0016] An embodiment of the invention is illustrated by a mesylate salt3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.

[0017] Another embodiment is a chloride salt3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.

[0018] Also included in the scope of the invention is the mesylate saltof3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-oneaccording to in crystalline form characterized by an X-ray powderdiffraction pattern having diffraction angles of: 7.39, 8.20, 9.03,9.90, 10.94, 15.45, 17.12, 17.84, 18.29, 18.64, 19.24, 19.77, 20.28,21.73, 22.49, 23.27, 24.15, 24.73, 25.40, 26.79, and 27.50.

[0019] A further embodiment is the mesylate salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-onein crystalline form characterized by an X-ray powder diffraction patternhaving diffraction angles of: 6.94, 8.01, 9.74, 10.47, 10.77, 11.75,12.61, 14.02, 15.28, 15.86, 16.93, 17.61, 18.69, 19.04, 19.47, 20.11,21.56, 21.94, 22.53, 23.85, and 27.22.

[0020] Another embodiment is the chloride salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-onein crystalline form characterized by an X-ray powder diffraction patternhaving diffraction angles of: 7.08, 7.86, 8.99, 14.54, 15.40, 16.14,16.81, 18.06, 19.91, 20.72, 22.72, 24.11, 26.09, 28.67, and 29.89.

[0021] And yet another embodiment is the chloride salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-onein crystalline form characterized by an X-ray powder diffraction patternhaving multiple diffraction peaks between 5° and 30° 2-theta and amelting endotherm of 284.08° C. at a rate of 10° C. per minute.

[0022] Also included in the scope of the invention is a mesylate salt of3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.

[0023] And yet another embodiment is a chloride salt of3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.

[0024] And still another embodiment is a mesylate salt of3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one.

[0025] A further embodiment is a chloride salt of3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one.

[0026] Another embodiment is the chloride salt3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-onein crystalline characterized by a reversible endotherm at 235° C. at ascan rate of 10° C. per minute.

[0027] And a still another embodiment is a mesylate salt of3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one.

[0028] A further embodiment is the mesylate salt of3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-onein crystalline form characterized by multiple reversible endotherms at82° C., 151.4° C., and 229° C. at a scan rate of 10° C. per minute.

[0029] And another ambodiment is a mesylate or chloride salt of

[0030]3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one;

[0031]3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one;

[0032]3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one;or

[0033] 3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1 H)-one.

[0034] Also included within the scope of the claims is a pharmaceuticalcomposition which is comprised of a salt of the present invention and apharmaceutically acceptable carrier. The present invention alsoencompasses a method of treating or preventing cancer in a mammal inneed of such treatment which is comprised of administering to saidmammal a therapeutically effective amount of a presently disclosed salt.Preferred cancers for treatment are selected from cancers of the brain,genitourinary tract, lymphatic system, stomach, larynx and lung. Anotherset of preferred forms of cancer are histiocytic lymphoma, lungadenocarcinoma, small cell lung cancers, pancreatic cancer, gioblastomasand breast carcinoma.

[0035] Also included is a method of treating or preventing a disease inwhich angiogenesis is implicated, which is comprised of administering toa mammal in need of such treatment a therapeutically effective amount ofa salt of Formula I. Such a disease in which angiogenesis is implicatedis ocular diseases such as retinal vascularization, diabeticretinopathy, age-related macular degeneration, and the like.

[0036] Also included within the scope of the present invention is amethod of treating or preventing inflammatory diseases which comprisesadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a salt of Formula I. Examples of such inflammatorydiseases are rheumatoid arthritis, psoriasis, contact dermatitis,delayed hypersensitivity reactions, and the like.

[0037] Also included is a method of treating or preventing a tyrosinekinase-dependent disease or condition in a mammal which comprisesadministering to a mammalian patient in need of such treatment atherapeutically effective amount of a salt of Formula I. The therapeuticamount varies according to the specific disease and is discernable tothe skilled artisan without undue experimentation.

[0038] A method of treating or preventing retinal vascularization whichis comprised of administering to a mammal in need of such treatment atherapeutically effective amount of a salt of Formula I is alsoencompassed by the present invention. Methods of treating or preventingocular diseases, such as diabetic retinopathy and age-related maculardegeneration, are also part of the invention. Also included within thescope of the present invention is a method of treating or preventinginflammatory diseases, such as rheumatoid arthritis, psoriasis, contactdermatitis and delayed hypersensitivity reactions, as well as treatmentor prevention of bone associated pathologies selected from osteosarcoma,osteoarthritis, and rickets.

[0039] The invention also contemplates the use of the instantly claimedsalts in combination with a second compound selected from:

[0040] 1) an estrogen receptor modulator,

[0041] 2) an androgen receptor modulator,

[0042] 3) retinoid receptor modulator,

[0043] 4) a cytotoxic agent,

[0044] 5) an antiproliferative agent,

[0045] 6) a prenyl-protein transferase inhibitor,

[0046] 7) an HMG-CoA reductase inhibitor,

[0047] 8) an HIV protease inhibitor,

[0048] 9) a reverse transcriptase inhibitor, and

[0049] 10) another angiogenesis inhibitor.

[0050] Preferred angiogenesis inhibitors are selected from the groupconsisting of a tyrosine kinase inhibitor, an inhibitor ofepidermal-derived growth factor, an inhibitor of fibroblast-derivedgrowth factor, an inhibitor of platelet derived growth factor, an MMP(matrix metalloprotease) inhibitor, an integrin blocker, interferon-α,interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor,carboxyamidotriazole, combreta-statin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, and an antibody to VEGF. Preferred estrogen receptormodulators are tamoxifen and raloxifene.

[0051] Also included in the scope of the claims is a method of treatingcancer which comprises administering a therapeutically effective amountof a salt of Formula I in combination with radiation therapy and/or incombination with a compound selected from:

[0052] 1) an estrogen receptor modulator,

[0053] 2) an androgen receptor modulator,

[0054] 3) retinoid receptor modulator,

[0055] 4) a cytotoxic agent,

[0056] 5) an antiproliferative agent,

[0057] 6) a prenyl-protein transferase inhibitor,

[0058] 7) an HMG-CoA reductase inhibitor,

[0059] 8) an HIV protease inhibitor,

[0060] 9) a reverse transcriptase inhibitor, and

[0061] 10) another angiogenesis inhibitor.

[0062] And yet another embodiment of the invention is a method oftreating cancer which comprises administering a therapeuticallyeffective amount of a salt of Formula I in combination with paclitaxelor trastuzumab.

[0063] Also within the scope of the invention is a method of reducing orpreventing tissue damage following a cerebral ischemic event whichcomprises administering a therapeutically effective amount of a salt ofFormula I.

[0064] These and other aspects of the invention will be apparent fromthe teachings contained herein.

[0065] “Tyrosine kinase-dependent diseases or conditions” refers topathologic conditions that depend on the activity of one or moretyrosine kinases. Tyrosine kinases either directly or indirectlyparticipate in the signal transduction pathways of a variety of cellularactivities including proliferation, adhesion and migration, anddifferentiation. Diseases associated with tyrosine kinase activitiesinclude the proliferation of tumor cells, the pathologicneovascularization that supports solid tumor growth, ocularneovascularization (diabetic retinopathy, age-related maculardegeneration, and the like) and inflammation (psoriasis, rheumatoidarthritis, and the like).

[0066] The salts of the present invention may have asymmetric centers,chiral axes, and chiral planes (as described in: E. L. Eliel and S. H.Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York,1994, pages 1119-1190), and occur as racemates, racemic mixtures, and asindividual diastereomers, with all possible isomers and mixturesthereof, including optical isomers, being included in the presentinvention. In addition, the salts disclosed herein may exist astautomers and both tautomeric forms are intended to be encompassed bythe scope of the invention, even though only one tautomeric structure isdepicted. For example, any claim to compound A below is understood toinclude tautomeric structure B, and vice versa, as well as mixturesthereof.

UTILITY

[0067] The instant salts are useful as pharmaceutical agents formammals, especially for humans, in the treatment of tyrosine kinasedependent diseases. Such diseases include the proliferation of tumorcells, the pathologic neovascularization (or angiogenesis) that supportssolid tumor growth, ocular neovascularization (diabetic retinopathy,age-related macular degeneration, and the like) and inflammation(psoriasis, rheumatoid arthritis, and the like). Based onpharmacokinetic studies in animals, the prsently claimed salts have anunexpectedly superior oral activity profile compared to thecorresponding free base and are therefore particularly suited for oraladministration. They may, however, be adminsitered via other routes asdescribed herein.

[0068] The salts of the instant invention may be administered topatients for use in the treatment of cancer. The instant salts inhibittumor angiogenesis, thereby affecting the growth of tumors (J. Rak etal. Cancer Research, 55:4575-4580, 1995). The anti-angiogenesisproperties of the instant salts are also useful in the treatment ofcertain forms of blindness related to retinal vascularization.

[0069] The disclosed salts are also useful in the treatment of certainbone-related pathologies, such as osteosarcoma, osteoarthritis, andrickets, also known as oncogenic osteomalacia. (Hasegawa et al.,Skeletal Radiol., 28, pp.41-45, 1999; Gerber et al., Nature Medicine,Vol. 5, No. 6, pp.623-628, June 1999). And since VEGF directly promotesosteoclastic bone resorption through KDR/Flk-1 expressed in matureosteoclasts (FEBS Let. 473:161-164 (2000); Endocrinology, 141:1667(2000)), the instant salts are also useful to treat and preventconditions related to bone resorption, such as osteoporosis and Paget'sdisease.

[0070] The claimed salts can also be used to reduce or prevent tissuedamage which occurs after cerebral ischemic events, such as stroke, byreducing cerebral edema, tissue damage, and reperfusion injury followingischemia. (Drug News Perspect 11:265-270 (1998); J. Clin. Invest.104:1613-1620 (1999).)

[0071] The salts of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers or diluents, optionally with knownadjuvants, such as alum, in a pharmaceutical composition, according tostandard pharmaceutical practice.

[0072] For oral use of a chemotherapeutic compound according to thisinvention, the compound may be administered, for example, in the form oftablets or capsules, or as an aqueous solution or suspension. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch, and lubricating agents, such as magnesiumstearate, are commonly added. For oral administration in capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents may be added.

[0073] The salts of the instant invention may also be co-administeredwith other well known therapeutic agents that are selected for theirparticular usefulness against the condition that is being treated. Forexample, in the case of bone-related disorders, combinations that wouldbe useful include those with antiresorptive bisphosphonates, such asalendronate and risedronate; integrin blockers (defined further below),such as α_(v)β₃ antagonists; conjugated estrogens used in hormonereplacement therapy, such as PREMPRO®, PREMARIN® and ENDOMETRION®;selective estrogen receptor modulators (SERMs), such as raloxifene,droloxifene, CP-336,156 (Pfizer) and lasofoxifene; cathespin Kinhibitors; and ATP proton pump inhibitors.

[0074] The instant salts are also useful in combination with knownanti-cancer agents. Such known anti-cancer agents include the following:estrogen receptor modulators, androgen receptor modulators, retinoidreceptor modulators, cytotoxic agents, antiproliferative agents,prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors, HIVprotease inhibitors, reverse transcriptase inhibitors, and otherangiogenesis inhibitors.

[0075] “Estrogen receptor modulators” refers to compounds whichinterfere or inhibit the binding of estrogen to the receptor, regardlessof mechanism. Examples of estrogen receptor modulators include, but arenot limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081 ,toremifene, fulvestrant,4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate,4,4′-dihydroxybenzophenone-2,4-dinitrophenylydrazone, and SH646.

[0076] “Androgen receptor modulators” refers to compounds whichinterfere or inhibit the binding of androgens to the receptor,regardless of mechanism. Examples of androgen receptor modulatorsinclude finasteride and other 5α-reductase inhibitors, nilutamide,flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0077] “Retinoid receptor modulators” refers to compounds whichinterfere or inhibit the binding of retinoids to the receptor,regardless of mechanism. Examples of such retinoid receptor modulatorsinclude bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, α-difluoromethylomithine, ILX23-7553,trans-N-(4′-hydroxyphenyl)retinamide, and N-4-carboxyphenyl retinamide.

[0078] “Cytotoxic agents” refer to compounds which cause cell deathprimarily by interfering directly with the cell's functioning or inhibitor interfere with cell myosis, including alkylating agents, tumornecrosis factors, intercalators, microtubulin inhibitors, andtopoisomerase inhibitors.

[0079] Examples of cytotoxic agents include, but are not limited to,tirapazimine, sertenef, cachectin, ifosfamide, tasonermin, lonidamine,carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine,fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin,estramustine, improsulfan tosilate, trofosfamide, nimustine,dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin,cisplatin, irofulven, dexifosfamide,cis-aminedichloro(2-methylyridine)platinum, benzylguanine, glufosfamide,GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine,arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin,amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin.

[0080] Examples of microtubulin inhibitors include paclitaxel, vindesinesulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol,rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin,RPR109881, BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)enzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide,TDX258, and BMS188797.

[0081] Some examples of topoisomerase inhibitors are topotecan,hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:b,7]indolizino[1,2b]quinoline-10,13(9H,15H)dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide,asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]phenanthridinium,6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide,N-(2-(dimethylamino)ethyl)acridine-4-carboxamide,6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one,and dimesna.

[0082] “Antiproliferative agents” includes antisense RNA and DNAoligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001,and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin,doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine,cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed,nelzarabine, 2′-deoxy-2′-methylidenecytidine,2′-fluoromethylene-2′-deoxycytidine,N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl) urea,N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine,aplidine, ecteinascidin, troxacitabine,4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin,5-flurouracil, alanosine,11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)tetradeca-2,4,6-trien-9-ylacetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase,2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, and3-aminopyridine-2-carboxaldehyde thiosemicarbazone. “Antiproliferativeagents” also includes monoclonal antibodies to growth factors, otherthan those listed under “angiogenesis inhibitors”, such as trastuzumab,and tumor suppressor genes, such as p53, which can be delivered viarecombinant virus-mediated gene transfer (see U.S. Pat. No. 6,069,134,for example).

[0083] “HMG-CoA reductase inhibitors” refers to inhibitors of3-hydroxy-3-methylglutaryl-CoA reductase. Compounds which haveinhibitory activity for HMG-CoA reductase can be readily identified byusing assays well-known in the art. For example, see the assaysdescribed or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131at pp. 30-33. The terms “HMG-CoA reductase inhibitor” and “inhibitor ofHMG-CoA reductase” have the same meaning when used herein.

[0084] Examples of HMG-CoA reductase inhibitors that may be used includebut are not limited to lovastatin (MEVACOR®; see U.S. Pat. Nos.4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Pat.Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; seeU.S. Pat. Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and5,180,589), fluvastatin (LESCOL®; see U.S. Pat. Nos. 5,354,772,4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896),atorvastatin (LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893,5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin andBAYCHOL®; see U.S. Pat. No. 5,177,080). The structural formulas of theseand additional HMG-CoA reductase inhibitors that may be used in theinstant methods are described at page 87 of M. Yalpani, “CholesterolLowering Drugs”, Chemistry & Industry, pp. 85-89 (Feb. 5, 1996) and U.S.Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitoras used herein includes all pharmaceutically acceptable lactone andopen-acid forms (i.e., where the lactone ring is opened to form the freeacid) as well as salt and ester forms of compounds which have HMG-CoAreductase inhibitory activity, and therefor the use of such salts,esters, open-acid and lactone forms is included within the scope of thisinvention. An illustration of the lactone portion and its correspondingopen-acid form is shown below as structures I and II.

[0085] In HMG-CoA reductase inhibitors where an open-acid form canexist, salt and ester forms may preferably be formed from the open-acid,and all such forms are included within the meaning of the term “HMG-CoAreductase inhibitor” as used herein. Preferably, the HMG-CoA reductaseinhibitor is selected from lovastatin and simvastatin, and mostpreferably simvastatin. Herein, the term “pharmaceutically acceptablesalts” with respect to the HMG-CoA reductase inhibitor shall meannon-toxic salts of the compounds employed in this invention which aregenerally prepared by reacting the free acid with a suitable organic orinorganic base, particularly those formed from cations such as sodium,potassium, aluminum, calcium, lithium, magnesium, zinc andtetramethylammonium, as well as those salts formed from amines such asammonia, ethylenediamine, N-methylglucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine,1-p-chlorobenzyl-2-pyrrolidine-1′-yl-methylbenzimidazole, diethylamine,piperazine, and tris(hydroxymethyl) aminomethane. Further examples ofsalt forms of HMG-CoA reductase inhibitors may include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate,estolate, esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynapthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote,palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, triethiodide, and valerate.

[0086] Ester derivatives of the described HMG-CoA reductase inhibitorcompounds may act as prodrugs which, when absorbed into the bloodstreamof a warm-blooded animal, may cleave in such a manner as to release thedrug form and permit the drug to afford improved therapeutic efficacy.

[0087] “Prenyl-protein transferase inhibitor” refers to a compound whichinhibits any one or any combination of the prenyl-protein transferaseenzymes, including farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). Examples of prenyl-protein transferase inhibiting compoundsinclude (±)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(−)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,(+)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone,5(S)-n-butyl-1-(2,3-dimethylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,(S)-1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl)methyl)-2-piperazinone,5(S)-n-Butyl-1-(2-methylphenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone,1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-2-methyl-5-imidazolylmethyl]-2-piperazinone,1-(2,2-diphenylethyl)-3-[N-(1-(4-cyanobenzyl)-1H-imidazol-5-ylethyl)carbamoyl]piperidine,4-{5-[4-Hydroxymethyl-4-(4-chloropyridin-2-ylmethyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methylimidazol-1-ylmethyl}benzonitrile,4-{3-[4-(2-oxo-2H-pyridin-1-yl)benzyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(5-chloro-2-oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-{3-[4-(2-Oxo-2H-[1,2′]bipyridin-5′-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile,4-[3-(2-Oxo-1-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile,18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1H-imidazo[4,3-c][1,11,4]dioxaazacyclo-nonadecine-9-carbonitrile,(±)-19,20-Dihydro-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile,19,20-dihydro-19-oxo-5H,17H-18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-carbonitrile,and(+)-19,20-Dihydro-3-methyl-19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.

[0088] Other examples of prenyl-protein transferase inhibitors can befound in the following publications and patents: WO 96/30343, WO97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO98/29119, WO 95/32987, U.S. Pat. No. 5,420,245, U.S. Pat. No. 5,523,430,U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No.5,589,485, U.S. Pat. No. 5,602,098, European Patent Publ. 0 618 221,European Patent Publ. 0 675 112, European Patent Publ. 0 604 181,European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917,WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example ofthe role of a prenyl-protein transferase inhibitor on angiogenesis seeEuropean J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).

[0089] Examples of IV protease inhibitors include amprenavir, abacavir,CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir,ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Examples ofreverse transcriptase inhibitors include delaviridine, efavirenz,GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddI.

[0090] “Angiogenesis inhibitors” refers to compounds that inhibit theformation of new blood vessels, regardless of mechanism. Examples ofangiogenesis inhibitors include, but are not limited to, tyrosine kinaseinhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1(VEGFR1) and Flk-1/KDR (VEGFR20), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, interferon-α,interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors,including nonsteroidal anti-inflammatories (NSAIDs) like aspirin andibuprofen as well as selective cyclo-oxygenase-2 inhibitors likecelecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69,p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec.,Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin,Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107(1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol.57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)),carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab.Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, NatureBiotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature,362, 841-844 (1993)).

[0091] Other examples of angiogenesis inhibitors include, but are notlimited to, endostation, ukrain, ranpirnase, IM862,5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfatedmannopentaose phosphate,7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone(SU5416).

[0092] As used above, “integrin blockers” refers to compounds whichselectively antagonize, inhibit or counteract binding of a physiologicalligand to the α_(v)β₃ integrin, to compounds which selectivelyantagonize, inhibit or counteract binding of a physiological ligand tothe αvβ5 integrin, to compounds which antagonize, inhibit or counteractbinding of a physiological ligand to both the α_(v)β₃ integrin and theα_(v)β₅ integrin, and to compounds which antagonize, inhibit orcounteract the activity of the particular integrin(s) expressed oncapillary endothelial cells. The term also refers to antagonists of theα_(v)β₆, α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins. The termalso refers to antagonists of any combination of α_(v)β₃, α_(v)β₅,α_(v)β₆, α_(v)β₈, α₁β₁, α₂β₁, α₅β₁, α₆β₁ and α₆β₄ integrins.

[0093] Some specific examples of tyrosine kinase inhibitors includeN-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one,17-(allylamino)-17-demethoxygeldanamycin,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline,N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine,BIBX1382,2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein,STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate,4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A,N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

[0094] The instantly claimed salts are also useful, alone or incombination with platelet fibrinogen receptor (GP IIb/IIIa) antagonists,such as tirofiban, to inhibit metastasis of cancerous cells. Tumor cellscan activate platelets largely via thrombin generation. This activationis associated with the release of VEGF. The release of VEGF enhancesmetastasis by increasing extravasation at points of adhesion to vascularendothelium (Amirkhosravi, Platelets 10, 285-292, 1999). Therefore, thepresent compounds can serve to inhibit metastasis, alone or incombination with GP IIb/IIIa) antagonists. Examples of other fibrinogenreceptor antagonists include abciximab, eptifibatide, sibrafiban,lamifiban, lotrafiban, cromofiban, and CT50352.

[0095] If formulated as a fixed dose, such combination products employthe salts of this invention within the dosage range described below andthe other pharmaceutically active agent(s) within its approved dosagerange. Compounds of the instant invention may alternatively be usedsequentially with known pharmaceutically acceptable agent(s) when acombination formulation is inappropriate.

[0096] The term “administration” and variants thereof (e.g.,“administering” a compound) in reference to a compound of the inventionmeans introducing the compound or a prodrug of the compound into thesystem of the animal in need of treatment. When a compound of theinvention or prodrug thereof is provided in combination with one or moreother active agents (e.g., a cytotoxic agent, etc.), “administration”and its variants are each understood to include concurrent andsequential introduction of the compound or prodrug thereof and otheragents.

[0097] As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

[0098] The term “therapeutically effective amount” as used herein meansthat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician.

[0099] The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

[0100] The present invention also encompasses a pharmaceuticalcomposition useful in the treatment of cancer, comprising theadministration of a therapeutically effective amount of the salts ofthis invention, with or without pharmaceutically acceptable carriers ordiluents. Suitable compositions of this invention include aqueoussolutions comprising compounds of this invention and pharmacologicallyacceptable carriers, e.g., saline, at a pH level, e.g., 7.4.

[0101] When a compound according to this invention is administered intoa human subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

[0102] In one exemplary application, a suitable amount of compound isadministered to a mammal undergoing treatment for cancer. Administrationoccurs in an amount between about 0.1 mg/kg of body weight to about 60mg/kg of body weight per day, preferably of between 0.5 mg/kg of bodyweight to about 40 mg/kg of body weight per day.

ASSAYS

[0103] The compounds of the instant invention described in the Exampleswere tested by the assays described below and were found to have kinaseinhibitory activity. Other assays are known in the literature and couldbe readily performed by those of skill in the art (see, for example,Dhanabal et al., Cancer Res. 59:189-197; Xin et al., J. Biol. Chem.274:9116-9121; Sheu et al., Anticancer Res. 18:4435-4441; Ausprunk etal., Dev. Biol. 38:237-248; Gimbrone et al., J. Natl. Cancer Inst.52:413-427; Nicosia et al., In Vitro 18:538-549).

[0104] I. VEGF Receptor Kinase Assay

[0105] VEGF receptor kinase activity is measured by incorporation ofradio-labeled phosphate into polyglutamic acid, tyrosine, 4:1 (pEY)substrate. The phosphorylated pEY product is trapped onto a filtermembrane and the incorporation of radio-labeled phosphate quantified byscintillation counting.

MATERIALS

[0106] VEGF Receptor Kinase

[0107] The intracellular tyrosine kinase domains of human KDR (Terman,B. I. et al. Oncogene (1991) vol. 6, pp. 1677-1683.) and Flt-1 (Shibuya,M. et al. Oncogene (1990) vol. 5, pp. 519-524) were cloned asglutathione S-transferase (GST) gene fusion proteins. This wasaccomplished by cloning the cytoplasmic domain of the KDR kinase as anin frame fusion at the carboxy terminus of the GST gene. Solublerecombinant GST-kinase domain fusion proteins were expressed inSpodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen).

[0108] The other materials used and their compositions were as follows:

[0109] Lysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,0.5% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin, pepstatinand aprotinin and 1 mM phenylmethylsulfonyl fluoride (all Sigma).

[0110] Wash buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM EDTA,0.05% triton X-100, 10% glycerol, 10 mg/mL of each leupeptin, pepstatinand aprotinin and 1 mM phenylmethylsulfonyl fluoride.

[0111] Dialysis buffer: 50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mMEDTA, 0.05% triton X-100, 50% glycerol, 10 mg/mL of each leupeptin,pepstatin and aprotinin and 1 mM phenylmethylsulfonyl fluoride.

[0112] 10×reaction buffer: 200 mM Tris, pH 7.4, 1.0 M NaCl, 50 mM MnCl₂,10 mM DTT and 5 mg/mL bovine serum albumin (Sigma).

[0113] Enzyme dilution buffer: 50 mM Tris, pH 7.4, 0.1 M NaCl, 1 mM DTT,10% glycerol, 100 mg/mL BSA.

[0114] 10×Substrate: 750 μg/mL poly (glutamic acid, tyrosine; 4:1)(Sigma).

[0115] Stop solution: 30% trichloroacetic acid, 0.2 M sodiumpyrophosphate (both Fisher).

[0116] Wash solution: 15% trichloroacetic acid, 0.2 M sodiumpyrophosphate.

[0117] Filter plates: Millipore #MAFC NOB, GF/C glass fiber 96 wellplate.

METHOD

[0118] A. Protein Purification

[0119] 1. Sf21 cells were infected with recombinant virus at amultiplicity of infection of 5 virus particles/cell and grown at 27° C.for 48 hours.

[0120] 2. All steps were performed at 4° C. Infected cells wereharvested by centrifugation at 1000×g and lysed at 4° C. for 30 minuteswith {fraction (1/10)} volume of lysis buffer followed by centrifugationat 100,000×g for 1 hour. The supernatant was then passed over aglutathione Sepharose column (Pharmacia) equilibrated in lysis bufferand washed with 5 volumes of the same buffer followed by 5 volumes ofwash buffer. Recombinant GST-KDR protein was eluted with wash buffer/10mM reduced glutathione (Sigma) and dialyzed against dialysis buffer.

[0121] B. VEGF Receptor Kinase Assay

[0122] 1. Add 5 μl of inhibitor or control to the assay in 50% DMSO.

[0123] 2. Add 35 μl of reaction mix containing 5 μl of 10×reactionbuffer, 5 μl 25 mM ATP/10 μCi [³³P]ATP (Amersham), and 5 μl10×substrate.

[0124] 3. Start the reaction by the addition of 10 μl of KDR (25 nM) inenzyme dilution buffer.

[0125] 4. Mix and incubate at room temperature for 15 minutes.

[0126] 5. Stop by the addition of 50 μl stop solution.

[0127] 6. Incubate for 15 minutes at 4° C.

[0128] 7. Transfer a 90 μl aliquot to filter plate.

[0129] 8. Aspirate and wash 3 times with wash solution.

[0130] 9. Add 30 μl of scintillation cocktail, seal plate and count in aWallac Microbeta scintillation counter.

[0131] II. Human Umbilical Vein Endothelial Cell Mitogenesis Assay

[0132] Human umbilical vein endothelial cells (HUVECs) in cultureproliferate in response to VEGF treatment and can be used as an assaysystem to quantify the effects of KDR kinase inhibitors on VEGFstimulation. In the assay described, quiescent HUVEC monolayers aretreated with vehicle or test compound 2 hours prior to addition of VEGFor basic fibroblast growth factor (bFGF). The mitogenic response to VEGFor bFGF is determined by measuring the incorporation of [³H]thymidineinto cellular DNA.

MATERIALS

[0133] HUVECs: HUVECs frozen as primary culture isolates are obtainedfrom Clonetics Corp. Cells are maintained in Endothelial Growth Medium(EGM; Clonetics) and are used for mitogenic assays described in passages1-5 below.

[0134] Culture Plates: NUNCLON 96-well polystyrene tissue culture plates(NUNC #167008).

[0135] Assay Medium: Dulbecco's modification of Eagle's mediumcontaining 1 mg/mL glucose (low-glucose DMEM; Mediatech) plus 10% (v/v)fetal bovine serum (Clonetics).

[0136] Test Compounds: Working stocks of test compounds are dilutedserially in 100% dimethylsulfoxide (DMSO) to 400-fold greater than theirdesired final concentrations. Final dilutions to 1×concentration aremade directly into Assay Medium immediately prior to addition to cells.

[0137] 10×Growth Factors: Solutions of human VEGF₁₆₅ (500 ng/mL; R&DSystems) and bFGF (10 ng/mL; R&D Systems) are prepared in Assay Medium.

[0138] 10×[³H]Thymidine: [Methyl-³H]thymidine (20 Ci/mmol; Dupont-NEN)is diluted to 80 μCi/mL in low-glucose DMEM.

[0139] Cell Wash Medium: Hank's balanced salt solution (Mediatech)containing 1 mg/mL bovine serum albumin (Boehringer-Mannheim).

[0140] Cell Lysis Solution: 1 N NaOH, 2% (w/v) Na₂CO₃.

METHOD

[0141] 1. HUVEC monolayers maintained in EGM are harvested bytrypsinization and plated at a density of 4000 cells per 100 μL AssayMedium per well in 96-well plates. Cells are growth-arrested for 24hours at 37° C. in a humidified atmosphere containing 5% CO₂.

[0142] 2. Growth-arrest medium is replaced by 100 μL Assay Mediumcontaining either vehicle (0.25% [v/v] DMSO) or the desired finalconcentration of test compound. All determinations are performed intriplicate. Cells are then incubated at 37° C. with 5% CO₂ for 2 hoursto allow test compounds to enter cells.

[0143] 3. After the 2-hour pretreatment period, cells are stimulated byaddition of 10 μL/well of either Assay Medium, 10×VEGF solution or10×bFGF solution. Cells are then incubated at 37° C. and 5% CO₂.

[0144] 4. After 24 hours in the presence of growth factors,10×[³H]thymidine (10 μL/well) is added.

[0145] 5. Three days after addition of [³H]thymidine, medium is removedby aspiration, and cells are washed twice with Cell Wash Medium (400μL/well followed by 200 μL/well). The washed, adherent cells are thensolubilized by addition of Cell Lysis Solution (100 μL/well) and warmingto 37° C. for 30 minutes. Cell lysates are transferred to 7-mL glassscintillation vials containing 150 μL of water. Scintillation cocktail(5 mL/vial) is added, and cell-associated radioactivity is determined byliquid scintillation spectroscopy.

[0146] Based upon the foregoing assays the compounds of the presentinvention are inhibitors of VEGF and thus are useful for the inhibitionof angiogenesis, such as in the treatment of ocular disease, e.g.,diabetic retinopathy and in the treatment of cancers, e.g., solidtumors. The instant compounds inhibit VEGF-stimulated mitogenesis ofhuman vascular endothelial cells in culture with IC₅₀ values between0.01-5.0 μM. These compounds may also show selectivity over relatedtyrosine kinases (e.g., FGFR1 and the Src family; for relationshipbetween Src kinases and VEGFR kinases, see Eliceiri et al., MolecularCell, Vol. 4, pp.915-924, December 1999).

[0147] III. Flt-1 Kinase Assay

[0148] Flt-1 was expressed as a GST fusion to the Flt-1 kinase domainand was expressed in baculovirus/insect cells. The following protocolwas employed to assay compounds for Flt-1 kinase inhibitory activity:

[0149] 1. Inhibitors were diluted to account for the final dilution inthe assay, 1:20.

[0150] 2. The appropriate amount of reaction mix was prepared at roomtemperature:

[0151] 10×Buffer (20 mM Tris pH 7.4/0.1 M NaCl/1 mM DTT final)

[0152] 0.1M MnCl₂ (5 mM final)

[0153] pEY substrate (75 μg/mL)

[0154] ATP/[³³P]ATP (2.5 μM/1 μCi final)

[0155] BSA (500 μg/mL final).

[0156] 3. 5 μL of the diluted inhibitor was added to the reaction mix.(Final volume of 5 μL in 50% DMSO). To the positive control wells, blankDMSO (50%) was added.

[0157] 4. 35 μL of the reaction mix was added to each well of a 96 wellplate.

[0158] 5. Enzyme was diluted into enzyme dilution buffer (kept at 4°C.).

[0159] 6. 10 μL of the diluted enzyme was added to each well and mix (5nM final). To the negative control wells, 10 μL 0.5 M EDTA was added perwell instead (final 100 mM).

[0160] 7. Incubation was then carried out at room temperature for 30minutes.

[0161] 8. Stopped by the addition of an equal volume (50 μL) of 30%TCA/0.1M Na pyrophosphate.

[0162] 9. Incubation was then carried out for 15 minutes to allowprecipitation.

[0163] 10. Transfered to Millipore filter plate.

[0164] 11. Washed 3×with 15% TCA/0.1 M Na pyrophosphate (125 μL perwash).

[0165] 12. Allowed to dry under vacuum for 2-3 minutes.

[0166] 13. Dryed in hood for ˜20 minutes.

[0167] 14. Assembled Wallac Millipore adapter and added 50 μL ofscintillant to each well and counted.

[0168] IV. Flt-3 Kinase Assay

[0169] Flt-3 was expressed as a GST fusion to the Flt-3 kinase domain,and was expressed in baculovirus/insect cells. The following protocolwas employed to assay compounds for Flt-3 kinase inhibitory activity:

[0170] 1. Dilute inhibitors (account for the final dilution into theassay, 1:20)

[0171] 2. Prepare the appropriate amount of reaction mix at roomtemperature.

[0172] 10×Buffer (20 mM Tris pH 7.4/0.1 M NaCl/1 mM DTT final)

[0173] 0.1M MnCl₂ (5 mM final)

[0174] pEY substrate (75 μg/mL)

[0175] ATP/[³³P]ATP (0.5 μM/L μCi final)

[0176] BSA (500 μg/mL final)

[0177] 3. Add 5 μL of the diluted inhibitor to the reaction mix. (Finalvolume of 5 μL in 50% DMSO). Positive control wells—add blank DMSO(50%).

[0178] 4. Add 35 μL of the reaction mix to each well of a 96 well plate.

[0179] 5. Dilute enzyme into enzyme dilution buffer (keep at 4° C.).

[0180] 6. Add 10 μL of the diluted enzyme to each well and mix (5-10 nMfinal). Negative control wells—add 10 μL 0.5 M EDTA per well instead(final 100 mM)

[0181] 7. Incubate at room temperature for 60 minutes.

[0182] 8. Stop by the addition of an equal volume (50 μL) of 30%TCA/0.1M Na pyrophosphate.

[0183] 9. Incubate for 15 minutes to allow precipitation.

[0184] 10. Transfer to Millipore filter plate.

[0185] 11. Wash 3×with 15% TCA/0.1M Na pyrophosphate (125 μL per wash).

[0186] 12. Allow to dry under vacuum for 2-3 minutes.

[0187] 13. Dry in hood for ˜20 minutes.

[0188] 14. Assemble Wallac Millipore adapter and add 50 μL ofscintillant to each well and count.

EXAMPLES

[0189] Examples provided are intended to assist in a furtherunderstanding of the invention. Particular materials employed, speciesand conditions are intended to be illustrative of the invention and notlimiting of the reasonable scope thereof.

[0190] The free bases used to prepare the salts of this invention may beobtained by employing the procdures described below as well as thosedisclosed in WO 01/29025, published Apr. 26, 2001, hereby incorporatedby reference. In addition, other procedures may be used by standardmanipulations of reactions that are known in the literature.

[0191] HPLC Methods Used Isocratic method (for solubility studies)Column: BDS HYPESIL, C18 (250 mm × 46 mm), 5 μm particle size ColumnTemperature: ambient Detector: 230 nm (UV wavelength) Column Temp.ambient Flow Rate: 1.0 mL/min Injection Volume: 20 μL Mobile Phase: A)0.1% Phosphoric Acid B) 100% Acetonitrile Diluent: 50% Acetonitrile-DIwater Gradient Profile: (A/B) starts from (60/40) and stays at (60/40)for 20 minutes. Run Time: 20 minutes

EXAMPLE 1

[0192] Salts of3-[5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one(1-11)

[0193] To a mechanically stirred solution of 1H-Indole-5-carboxylic acid(1-1, 20.01 g, 124 mmol) in THF (500 mL) was added at ambienttemperature slowly a solution of 1M-LAH in toluene (186 mL, 186 mmol,1.5 equiv). The reaction mixture was heated at reflux for 1 hour,quenched with ice, partitioned between ethylacetate and saturatedaqueous NaHCO₃. The organic layer was washed with brine, separated,dried (MgSO₄) and concentrated in vacuo. The crude product solidifiedupon standing under the reduced pressure. The crude solid was suspendedin hexanes (200 mL) and ethyl acetate (10 mL), stirred overnight,collected by filtration and air-dried to afford the desired product as alight brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.24 (br s, 1H), 7.62 (s,1H), 7.36 (d, 1H, J=8.4 Hz), 7.23 (d, 1H, J=8.4 Hz), 7.20 (s, 1H), 6.54(s, 1H), 4.75 (s, 2H), 1.68 (s, 1H).

[0194] 5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (1-3)

[0195] A stirred solution of (1H-Indol-5-yl)-methanol (1-2, 16.5 g,112.1 mmol) in dichloromethane (300 mL) was subsequently treated atambient temperature with diisopropylethylamine (39 mL, 224.2 mmol, 2equiv), tert-butyldimethylsilyl chloride (18.6 g, 123.3 mmol, 1.1equiv), and 4-(N,N-dimethylamino)pyridine (1.37 g, 11.2 mmol, 0.1equiv). The reaction mixture was stirred at room temperature for 30minutes, concentrated in vacuo, partitioned between ethyl acetate and0.5N-HCl. The organic layer was washed with brine, separated, dried(MgSO₄), concentrated in vacuo to give the crude silylether as a lightbrown solid. The crude product and di-tert-butyl dicarbonate (26.9,123.3 mmol) were dissolved in dichrolomethane (300 mL) and stirred atambient temperature in the presence of 4-(N,N-dimethylamino) pyridine(1.37 g, 11.2 mmol) for 2 hours. The reaction mixture was concentratedin vacuo, partitioned between ethyl acetate and 0.5N-HCl. The organiclayer was washed with brine, separated, dried (MgSO₄) and concentratedin vacuo to give the crude oil. Chromatography (SiO₂, 10% ethyl acetatein hexanes) afforded5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (1-3) as a white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.97(d, 1H, J=8.0 Hz), 7.47 (d, 1H, J=3.2 Hz), 7.41 (s, 1H), 7.15 (d, 1H,J=7.7 Hz), 6.44 (d, 1H, J=3.6 Hz), 4.72 (s, 2H), 1.56 (s, 9H), 0.84 (s,9H), 0.00 (s, 6H).

[0196]5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-tert-butyloxycarbonylindole-2-boronicacid (1-4)

[0197] To a stirred solution of5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (1-3, 38.6 g, 106.7 mmol) in tetrahydrofuran (400 mL)was slowly added at −78° C. a solution of lithiun diisopropylamide intetrahydrofuran (2M, 80.1 mL, 160.1 mmol, 1.5 equiv). The reactionmixture was stirred at the same temperature for 1 hour, treated withtrimethylborate, warmed up to ambient temperature, and partitionedbetween ethyl acetate and 0.5N-HCl. The organic layer was washed withbrine, separated, dried (MgSO₄) and concentrated in vacuo to give thecrude solid. Trituation of the crude product with hexanes followed byfiltration and air-drying afforded the desired boronic acid (1-4) as awhite powder. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, 1H, J=6.8 Hz), 7.54 (s,1H), 7.47 (s, 1H), 7.32 (d, 1H, J=6.8 Hz), 7.10 (s, 1H), 4.82 (s, 2H),1.74 (s, 9H), 0.95 (s, 9H), 0.11 (s, 6H).

[0198] 3-Iodo-1H-quinolin-2-one (1-6)

[0199] The 2-chloro-3-iodoquinoline (1-5, 30.0 g) was weighed into a 250mL flask and suspended in of 50% aqueous acetic acid (125 mL). Themixture was heated to 100° C. and allowed to reflux for 16 hours tocompletion by TLC analysis of the crude reaction mixture. The mixturewas allowed to cool to ambient temperature followed by dilution with 200mL of water. The resulting suspension of the desired product wasisolated by vacuum filtration follows by washing with water (50 mL). Thewater and traces of acetic acid were removed under vacuum for 5 hours toafford the desired quinolinone as a tan powder (1-6). ¹H NMR (500 MHz,CDCl₃) δ 12.13 (br s, 1H), 8.71 (s, 1H), 7.65 (d, 1H, J=7.5 Hz), 7.54(m, 1H), 7.31 (d, 1H, J=8.0 Hz), 7.20 (m, 1H).

[0200]5-Hydroxymethyl-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (1-8)

[0201] A stirred mixture of the iodoquinolinone (1-6, 10 g, 36.9 mmol, 1equiv), the boronic acid (1-4, 7.5 g, 18.45 mmol, 0.5 equiv), tetrakis(triphenyl-phosphine) palladium (1.71 g, 1.48 mmol, 0.04 equiv), andlithium chloride (4.69 g, 110.7 mmol, 3 equiv) in dioxane/2M-aqueousNa₂CO₃ was degassed and heated at 80° C. until the boronic acid is notdetected by thin layer chromatography. Additional boronic acid (0.2equiv at a time) was added to the reaction mixture until all theiodoquinolinone (1-6) was consumed completely (1.5 equivalent of theboronic acid, 1-4, in total, was required). The reaction mixture waspartitioned between ethyl acetate and saturated aqueous NaHCO₃. Theorganic layer was washed with brine, separated, dried (MgSO₄) andconcentrated in vacuo. The crude oil (1-7) was dissolved intetrahydrofuran (100 mL), transferred to the PEG bottle, treated at 0°C. with HF-pyridine (15 mL) and stirred for 1 hour at ambienttemperature. The reaction mixture was partitioned between ethyl acetateand saturated aqueous NaHCO₃. The organic layer was washed with brine,separated, dried (MgSO₄) and concentrated in vacuo. The crude solid wastrituated with ethyl acetate and hexanes, collected by filtration andair-dried to afford the desired product (1-8) as a light yellow solid;¹H NMR (500 MHz, DMSO-d₆) δ 12.1 (s, 1H), 8.07 (s, 1H), 8.03 (d, 1H,J=8.5 Hz), 7.74 (d, 1H, J=7.5 Hz), 7.55 (s, 1H), 7.52 (t, 1H, J=7.5 Hz),7.35 (d, 1H, J=8.5 Hz), 7.30 (d, 1H, J=7.5 Hz), 7.22 (t, 1H, J=7.5 Hz),6.77 (s, 1H), 5.21 (t, 1H, J=5.5 Hz), 4.60 (d, 2H, J=5.5 Hz), 1.35 (s,9H).

[0202] 5-Formyl-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (1-9)

[0203] The pre-activated MnO₂ (34.5 g, 15 equiv) and the alcohol (1-8,10.32 g, 1.0 equiv) were weighed into a 1-liter flask and suspended indry dichloromethane (500 mL). The reaction mixture was heated to 45° C.and was complete by thin layer chromatography after 1 hour. The mixturewas allowed to cool to ambient temperature and the manganese oxide(s)were removed by vacuum filtration. The resulting pad of oxides on thefilter were triturated with hot THF and the solvent filtered throughunder vacuum to remove any product from the oxides. The resultingfiltrate was concentrated in vacuo to afford the crude aldehyde as ayellow solid. The solid was triturated with methanol (10 mL) and ethylacetate (15 mL) followed by vacuum filtration to isolate the pureproduct. The light-yellow aldehyde was dried under vacuum (1-9). ¹H NMR(500 MHz, DMSO-d₆) δ 12.15 (s, 1H), 10.08 (s, 1H), 8.26 (d, 1H, J=1.5Hz), 8.24 (d, 1H, J=8.5 Hz), 8.15 (s, 1H), 7.90 (dd, 1H, J=8.5, 1.5 Hz),7.77 (d, 1H, J=7.5 Hz), 7.55 (m, 1H), 7.37 (d, 1H, J=8.5 Hz), 7.24 (m,1H), 7.01 (s, 1H).

[0204]5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (1-10)

[0205] To a stirred solution of the aldehyde (1-9, 2.01 g, 5.15 mmol, 1equiv) and N-methanesulfonylpiperazine acetic acid salt (4.62 g, 20.60mmol, 4 equiv) in dichloroethane (400 mL) was added at ambienttemperature acetic acid (1.2 mL). The reaction mixture was treated withsodium triacetoxyborohydride and stirred for 3 hours. The reactionstopped at 76% of conversion and treated with MgSO₄ and additional 1 gof the hydride. After further stirring for 1 hour the reaction wascomplete. The reaction mixture was partitioned between ethyl acetate andsaturated aqueous NaHCO₃. The organic layer was once again washed withsaturated aqueous NaHCO₃, and then with brine, separated, dried with(Na₂SO₄) and concentrated in vacuo. The crude solid was dissolved indimethylformamide and treated with the activated carbon. The filtratesolution (celite) was concentrated to syrup which was quickly trituatedwith methanol (100 mL). The resulting solid was collected by filtration,redissolved in dimethylformamide, concentrated to syrup, trituated withmethanol (100 mL), collected by filtration and vacuum-dried to give5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (1-10) as a white powder; ¹H NMR (500 MHz,DMSO-d₆) δ 12.06 (s, 1H), 8.06 (s, 1H), 8.04 (d, 1H, J=8.5 Hz), 7.74 (d,1H, J=8.0 Hz), 7.55 (s, 1H), 7.53 (dt, 1H, J=8.0, 1.5 Hz), 7.35 (d, 1H,J=8.5 Hz), 7.30 (dd, 1H, J=8.5, 1.5 Hz), 7.22 (t, 1H, J=7.5 Hz), 6.76(s, 1H), 3.62 (s, 2H), 3.16 (m, 4H), 2.87 (s, 3H), 2.48 (m, 4H), 1.35(s, 9H).

[0206]3-[5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one(1-11)

[0207] A mixture of5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (1-10, 1.02 g, 1.863 mmol), dimethylsulfide (1.2mL), water (0.6 mL) and TFA (40 mL) in dichloromethane (40 mL) wasstirred for 1.5 hours. The reaction mixture was concentrated in vacuo,partitioned between ethyl acetate and saturated aqueous NaHCO₃. Theorganic layer was washed with brine, separated, dried (Na₂SO₄), andconcentrated in vacuo. The resulting crude solid was purified byreverse-phase liquid chromatography (H₂O/CH₃CN gradient with 0.1% TFApresent) to give trifluoroacetic acid salt of 1-11. All the fractionscontaining the desired product was partitioned between ethyl acetate andsaturated aqueous NaHCO₃. The organic layer was washed with brine,separated, dried (Na₂SO₄), and concentrated in vacuo to give3-[5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one(1-11) as a bright yellow solid; ¹H NMR (500 MHz, DMSO-d₆) δ 12.07 (s,1H), 11.54 (s, 1H), 8.53 (s, 1H), 7.73 (d, 1H, J=7.5 Hz), 7.52 (t, 1H,J=7.5 Hz), 7.47-7.46 (m, 2H), 7.38 (d, 1H, J=8.5 Hz), 7.29 (br s, 1H),7.25 (t, 1H, J=7.5 Hz), 7.08 (d, 1H, J=9.0 Hz), 3.57 (s, 2H), 3.11 (m,4H), 2.87 (s, 3H), 2.48 (m, 4H).

[0208] Solubility of Free Bases

[0209] The solubility of 1-11 at room temperature was determined in0.05M aqueous buffers, water and several organic solvents. The resultsare tabulated in Tables I and II. TABLE I pH Solubility Profile of 1-11Buffer pH_(final) Solubility (mg/mL) 0.05 M Carbonate 11.15 0.077 0.05 MCarbonate 10.09 0.079 0.05 M Phosphate 8.86 0.073 0.05 M Phosphate 7.930.074 0.05 M Phosphate 6.96 0.071 0.05 M Citrate 6.01 0.071 0.05 MCitrate 5.07 0.070 0.05 M Citrate 4.15 0.073 0.05 M Citrate 3.23 0.0920.01 N HCl 2.45 0.40

[0210] TABLE II Solubility of 1-11 in several solvent systems and HPβCD.Solvent Solubility (mg/mL) Water 0.00003 Ethanol 0.019 Isopropanol0.0065 Acetonitrile 0.084 50% Aqueous Can 0.060 25% HPβCD¹ 0.168 25%HPβCD² 4.56 20% HPβCD² 3.57 15% HPβCD² 2.35 10% HPβCD² 1.74

[0211] As can be discerned from these tables, the free base has a verylow solubility in water.

[0212] Measurement of Salt Solubility

[0213] The solid free base was treated with a concentrated solution ofthe appropriate acid (1.05-1.1 molar equivalence) and suspended in waterfor several days. The pH and concentration of the compound inequilibrium with the solid phase was measured by HPLC (UV-Visdetection). This method overestimates the solubility of the salt due tothe 5-10% excess of the acid that is added in the preparation of thesalts. For the same reason, the pH's that are obtained are most likelylower than the pH of the salt in water. The results are shown below inTable III. TABLE III Salt Solubility Salt of 1-11 Solubility (mg/mL) pHMesylate 0.58 2.3 Tartrate 0.90 2.4 HCl 0.43 2.3 Citrate 0.11 2.5Acetate 0.091 3.3 HBr 0.20 2.9 Maleate 0.16 2.5 Sulfate 0.13 1.5Besylate 0.0038 2.4

[0214] Physical Properties of the Mesylate Salt of 1-11

[0215] The mesylate salt of 1-11 has molecular formula C₂₄H₂₈N₄O₆S₂ anda molecular weight of 532.642. Three forms of the mesylate salt of 1-11have been observed: one amorphous salt and two crystalline salts.Initially an amorphous solid was obtained as determined by opticalmicroscopy under plane polarized light.

[0216] The amorphous mesylate salt was recrystallized from 50:50Ethanol:THF to give a crystalline powder (Salt 1-11A). The X-ray powderdiffraction pattern (XRPD) of the recrystallized mesylate salt 1-11A(FIG. 2) is indicative of crystalline material with multiple diffractionpeaks between 5° and 30° 2-theta. DSC of this material from 20° C. to350° C. at a heating rate of 10° C./minute shows a sharp endotherm at266° C. which is attributed to melting. TGA of this material from 20° C.to 350° C. at a heating rate of 10° C./minute showed a weight loss of1.25% between 20° C. and 125° C., attributable to residual solvent. Thismesylate salt has a solubility in water of 0.173 mg/mL. TABLE IVSolubility of Salt 1-11A Solvent Solubility (mg/mL) Water 0.173Isopropanol 0.02 Acetonitrile 0.063

[0217] A second batch of crystalline mesylate salt (Salt 1-11B) wasobtained by the following procdeure. To a stirred suspension of 1-11(4.026 g, 9.22 mmol) in MeOH was slowly added at room temperature (RT)one equivalent of a 0.3M solution of methanesulfonic acid (30.73 mL).After all the solid had dissolved, the mixture was filtered into a flaskand concentrated under reduced pressure while cooled to about 10° C. Theresulting solid was suspended with 200 mL of ethylacetate, filtered, anddried to afford the mesylate salt. This salt was found to be crystallineby XRPD (FIG. 2). This crystalline form is different by XRPD than theone recrystallized from the amorphous salt in EtOH/THF (Salt 1-11A).This form has a lower solubility in water 0.09 mg/mL than Salt 1-11Asuggesting it is a more stable form of the mesylate salt. Thesolubilities of Salt 1-11B are summarized in Table V below: TABLE VSolubility of Salt 1-11B Solvent Solubility (mg/mL) Water 0.09Isopropanol 0.003 Acetonitrile 0.03

[0218] HCl Salt

[0219] Two crystalline salts of 1-11 have been identified: 1-11C and1-11D. The XRPD of the HCl salt 1-11C (FIG. 3) verify that it iscrystalline. DSC showed a melting endotherm at 284° C. The compoundcontains 5.4% moisture up to 150° C. as evidenced by TGA. This salt alsoseems to decompose on melting as seen by a sharp drop in weight atmelting.

[0220] HCl salt 1-11D is also crystalline as witnessed by the X-raypowder diffraction pattern of the material, having multiple diffractionpeaks between 5° and 30° 2-theta. DSC shows a melting endotherm of284.08° C. (rate of 10° C./min). The compound is birefringent underplane polarized light. It is needle-shaped particles of approximately5-25 micron. The solubility of salt 1-11D was measured in water andvarious organic solvents. Table VI below summarizes the solubility ofthe 1-11D suspended in various solvents for 7 days at room temperature.TABLE VI Solubility of HCi Salt 1-11D RT for 7 days Solubility Solvents(mg/mL) Water 0.62 Ethanol 0.13 Isopropanol 0.057 Aqueous 4.34 ETOHAqueous IPA 3.99

[0221] The X-ray powder diffraction data for 1-11D is summarized below:2-Theta° Angstrom Count % d = 13.07327 6.756 13.07327 1197 62.1 d =10.92179 8.089 10.92179 721 37.4 d = 8.87959 9.953 8.87959 971 50.4 d =7.32324 12.075 7.32324 1376 71.4 d = 6.88388 12.849 6.88388 1069 55.5 d= 6.44424 13.730 6.44424 1010 52.4 d = 6.16135 14.364 6.16135 853 44.3 d= 5.95917 14.854 5.95917 1056 54.8 d = 5.81946 15.212 5.81946 1481 76.9d = 5.51333 16.062 5.51333 1556 80.7 d = 5.42028 16.340 5.42028 100952.4 d = 5.27926 16.780 5.27926 1129 58.6 d = 5.13623 17.250 5.13623 72137.4 d = 4.84647 18.290 4.84647 1927 100.0 d = 4.69650 18.880 4.69650987 51.2 d = 4.63537 19.131 4.63537 1010 52.4 d = 4.49882 19.717 4.498821239 64.3 d = 4.36248 20.340 4.36248 681 35.3 d = 4.27994 20.737 4.279941764 91.5 d = 4.12084 21.547 4.12084 729 37.8 d = 3.97380 22.354 3.97380811 42.1 d = 3.86205 23.009 3.86205 553 28.7 d = 3.70294 24.013 3.702941012 52.5 d = 3.66870 24.240 3.66870 842 43.7 d = 3.53317 25.185 3.533171380 71.6 d = 3.48450 25.542 3.48450 1161 60.2 d = 3.31645 26.8603.31645 962 49.9 d = 3.21463 27.728 3.21463 485 25.2 d = 3.10080 28.7673.10080 826 42.9 d = 3.03001 29.454 3.03001 390 20.2 d = 2.98281 29.9312.98281 556 28.9 d = 2.95392 30.231 2.95392 675 35.0 d = 2.90366 30.7672.90366 602 31.2 d = 2.84488 31.419 2.84488 403 20.9 d = 2.75928 32.4202.75928 477 24.8 d = 2.70643 33.071 2.70643 472 24.5 d = 2.43879 36.8242.43879 345 17.9

EXAMPLE 2

[0222] Salts of3-[5-(4-Methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one2-1

[0223] 2-1 was prepared by simple modifications of the protocolsdescribed below to make 3-10.

[0224]¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (s, 1H), 11.53 (s, 1H), 8.52 (s,1H), 7.73 (d, 1H, J=7.5 Hz), 7.52 (dt, 1H, J=8.5, 1.0 Hz), 7.46 (d, 1H,J=9.0 Hz), 7.45 (s, 1H), 7.38 (d, 1H, J=8.0 Hz), 7.29 (s, 1H), 7.25 (t,1H, J=7.5 Hz), 7.08 (dd, 1H, J=8.0, 1.0 Hz), 3.61 (s, 2H), 3.42 (m, 2H),2.83 (s, 3H), 2.54-2.50 (m, 6H);

[0225] Mesylate salt 2-1A was prepared by simple modifications of theprotocols described below to make 3-10B.

[0226]4-Methyl-5-oxo-1-[2-(2-oxo-1.2-dihydro-quinolin-3-yl)-1H-indol-5-ylmethyl]-[1,4]diazepan-1-ium;methanesulfonate; methanesulfonate (2-1A)

[0227]¹H NMR (300 MHz, DMSO-d₆) δ 12.21 (s, 1H), 11.82 (s, 1H), 10.81(br s, 1H), 8.61 (s, 1H), 7.76 (s, 1H), 7.74 (d, 1H, J=8.5 Hz), 7.60 (d,1H, J=8.0 Hz), 7.54 (t, 1H, J=8.0), 7.40 (d, 1H, J=8.0 Hz), 7.39 (s,1H), 7.31 (dd, 1H, J=8.5, 1.5 Hz), 7.26 (t, 1H, J=7.5 Hz), 4.41 (m, 2H),4.04 (m, 1H), 3.47 (m, 3H), 3.24-3.08 (m, 3H), 2.87 (s, 3H), 2.55 (m,1H).

[0228] HCl salt 2-1B was prepared by simple modifications of theprotocols described below to make 3-10A.

[0229]4-Methyl-5-oxo-1-[2-(2-oxo-1,2-dihydro-quinolin-3-yl)-1H-indol-5-ylmethyl]-[1,4]diazepan-1-ium;methanesulfonate; chloride (2-1B)

[0230]¹H NMR (500 MHz, DMSO-d₆) δ 12.21 (s, 1H), 11.82 (s, 1H), 10.81(br s, 1H), 8.61 (s, 1H), 7.76 (s, 1H), 7.74 (d, 1H, J=8.5 Hz), 7.60 (d,1H, J=8.0 Hz), 7.54 (t, 1H, J=8.0), 7.40 (d, 1H, J=8.0 Hz), 7.39 (s,1H), 7.31 (dd, 1H, J=8.5, 1.5 Hz), 7.26 (t, 1H, J=7.5 Hz), 4.41 (m, 2H),4.04 (m, 1H), 3.47 (m, 3H), 3.24-3.08 (m, 3H), 2.87 (s, 3H), 2.55 (m,1H).

[0231] Characteristics of the Free Base

[0232] The free base 2-1 is a yellow powder. Examination under polarizedlight by optical microscopy revealed that the crystals are birefringentindicating crystallinity. The crystals appear plate-like with most ofthe particles below 10 microns. TGA analysis with a scan rate of 10°C./min to 350° C. shows the solid loses 0.51% weight up to 125° C. andloses weight more rapidly past 275° C. DSC analysis with a scan rate of10° C./min to 350° C. shows a reversible endotherm at 292° C. Theaqueous solubility was measured using HPLC to quantitate the compound insuspension. The solubility at room temperature is 0.028 mg/mL.

[0233] Mesylate Salt

[0234] The mesylate salt 2-1A is a yellow powder. Examination underpolarized light by optical microscopy shows that the solid is notbirefringent, indicating that it is amorphous. TGA analysis at a scanrate of 10° C./min to 350° C. shows the solid loses 2.74% weight up to125° C. and decomposes above 250° C. DSC analysis with a scan rate of10° C./min to 350° C. does not show any reversible endotherm confirmingthat the solid is amorphous. A broad non-reversible endotherm centeredaround 87° C. is attributed to loss of solvent/moisture. The aqueoussolubility was measured using HPLC to quantitate the compound. Theaqueous solubility at room temperature is greater than 9.89 mg/mL. Inthe time scale of the experiment (24 hours) no crystallization wasobserved.

[0235] HCl Salt

[0236] The HCl salt 2-1B was also a yellow powder. Examination underpolarized light by optical microscopy showed that the solid containedsome particles that were birefringent and particles that are notbirefringent. This indicates the presence of both amorphous andcrystalline material.. TGA analysis with a scan rate of 10° C./min to350° C. shows the solid loses 2.4% weight up to 125° C. and decomposesabove 250° C. DSC analysis at a scan rate of 10° C./min to 350° C. showsa reversible endotherm at 231° C. indicating the existence of somecrystalline material. The aqueous solubility was measured using HPLC toquantitate the compound. The aqueous solubility at room temperature isgreater than 10.21 mg/mL. In the time scale of the experiment (24 hours)the solid did not crystallize out of this highly concentrated solution.

EXAMPLE 3

[0237] Salts of3-{5-[4-(2-Hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one

[0238] To a mechanically stirred solution of 1H-Indole-5-carboxylic acid(3-1, 20.01 g, 124 mmol) in tetrahydrofuran (500 mL) was added atambient temperature slowly a solution of 1M-LAH in toluene (186 mL, 186mmol, 1.5 equiv). The reaction mixture was heated at reflux for 1 h,quenched with ice, partitioned between EA and saturated aqueous NaHCO₃.The organic layer was washed with brine, separated, dried (MgSO₄) ansconcentrated in vacuo. The crude product solidified upon standing underthe reduced pressure. The crude solid was suspended in hexanes (200 mL)and ethyl acetate (10 mL), stirred overnight, collected by filtrationand air-dried to afford the desired product as a light brown solid. ¹HNMR (400 MHz, CDCl₃) δ 8.24 (br s, 1H), 7.62 (s, 1H), 7.36 (d, 1H, J=8.4Hz), 7.23 (d, 1H, J=8.4 Hz), 7.20 (s, 1H), 6.54 (s, 1H), 4.75 (s, 2H),1.68 (s, 1H).

[0239] 5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (3-3)

[0240] A stirred solution of (1H-Indol-5-yl)-methanol (3-2, 16.5 g,112.1 mmol) in dichloromethane (300 mL) was subsequently treated atambient temperature with diisopropylethylamine (39 mL, 224.2 mmol, 2equiv), tert-butyldimethylsilyl chloride (18.6 g, 123.3 mmol, 1.1equiv), and 4-(N,N-dimethylamino)pyridine (1.37 g, 11.2 mmol, 0.1equiv). The reaction mixture was stirred at rt for 30 min, concentratedin vacuo, partitioned between ethyl acetate and 0.5N-HCl. The organiclayer was washed with brine, separated, dried (MgSO₄), concentrated invacuo to give the crude silylether as a light brown solid. The crudeproduct and di-tert-butyl dicarbonate (26.9, 123.3 mmol) were dissolvedin dichrolomethane (300 mL) and stirred at ambient temperature in thepresence of 4-(N,N-dimethylamino)pyridine (1.37 g, 11.2 mmol) for 2 h.The reaction mixture was concentrated in vacuo, partitioned betweenethyl acetate and 0.5N-HCl. The organic layer was washed with brine,separated, dried (MgSO₄) and concentrated in vacuo to give the crudeoil. Chromatography (SiO₂, 10% ethyl acetate in hexanes) afforded5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (3-3, 38.6 g, 95%) as a white solid; ¹H NMR (400 MHz,CDCl₃) δ 7.97 (d, 1H, J=8.0 Hz), 7.47 (d, 1H, J=3.2 Hz), 7.41 (s, 1H),7.15 (d, 1H, J=7.7 Hz), 6.44 (d, 1H, J=3.6 Hz), 4.72 (s, 2H), 1.56 (s,9H), 0.84 (s, 9H), 0.00 (s, 6H).

[0241]5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-tert-butyloxycarbonylindole-2-boronicacid (3-4)

[0242] To a stirred solution of5-(tert-Butyl-dimethyl-silanyloxymethyl)-indole-1-carboxylic acidtert-butyl ester (3-3, 38.6 g, 106.7 mmol) in tetrahydrofuran (400 mL)was slowly added at −78 deg C. a solution of lithiun diisopropylamide intetrahydrofuran (2M, 80.1 mL, 160.1 mmol, 1.5 equiv). The reactionmixture was stirred at the same temperature for 1 h, treated withtrimethylborate, warmed up to ambient temperature, and partitionedbetween ethyl acetate and 0.5N-HCl. The organic layer was washed withbrine, separated, dried (MgSO₄) and concentrated in vacuo to give thecrude solid. Trituration of the crude product with hexanes followed byfiltration and air-drying afforded the desired boronic acid (3-4, 41.3g, 95%) as a white powder; ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, 1H, J=6.8Hz), 7.54 (s, 1H), 7.47 (s, 1H), 7.32 (d, 1H, J=6.8 Hz), 7.10 (s, 1H),4.82 (s, 2H), 1.74 (s, 9H), 0.95 (s, 9H), 0.11 (s, 6H).

[0243] 3-Iodo-1H-quinolin-2-one (3-5)

[0244] The 2-chloro-3-iodoquinoline (30.0 g) was weighed into a 250 mLflask and suspended in of 50% aqueous acetic acid (125 mL). The mixturewas heated to 100 C and allowed to reflux for 16 h to completion by TLCanalysis of the crude reaction mixture. The mixture was allowed to coolto ambient temperature followed by dilution with 200 mL of water. Theresulting a suspension of the desired product was isolated by vacuumfiltration follows by washing with water (50 mL). The water and tracesof acetic acid were removed under vacuum for 5 h to afford the desiredquinolinone as a tan powder (5-5, 26.5 g, 94%); ¹H NMR (500 MHz, CDCl₃)δ 12.13 (br s, 1H), 8.71 (s, 1H), 7.65 (d, 1H, J=7.5 Hz), 7.54 (m, 1H),7.31 (d, 1H, J=8.0 Hz), 7.20 (m, 1H).

[0245]5-Hydroxymethyl-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (5-7)

[0246] A stirred mixture of the iodoquinolinone (5-5, 10 g, 36.9 mmol, 1equiv), the boronic acid (5-4, 7.5 g, 18.45 mmol, 0.5 equiv),tetrakis(triphenylphosphine)palladium (1.71 g, 1.48 mmol, 0.04 equiv),and lithium chloride (4.69 g, 110.7 mmol, 3 equiv) in dioxane/2M-aqueousNa₂CO₃ was degassed and heated at 80 deg C. until the boronic acid isnot detected by thin layer chromatography. Additional boronic acid (0.2equiv at a time) was added to the reaction mixture until all theiodoquinolinone (5-5) was consumed completely (1.5 equivalent of theboronic acid, 5-4, in total, was required). The reaction mixture waspartitioned between ethyl acetate and saturated aqueous NaHCO₃. Theorganic layer was washed with brine, separated, dried (MgSO₄) andconcentrated in vacuo. The crude oil (5-6) was dissolved intetrahydrofuran (100 mL), transferred to the PEG bottle, treated at 0deg C. with HF-pyridine (15 mL) and stirred for 1 h at ambienttemperature. The reaction mixture was partitioned between ethyl acetateand saturated aqueous NaHCO₃. The organic layer was washed with brine,separated, dried (MgSO₄) and concentrated in vacuo. The crude solid wastriturated with ethyl acetate and hexanes, collected by filtration andair-dried to afford the desired product (5-7) as a light yellow solid(12.4 g, 86%); ¹H NMR (500 MHz, DMSO-d₆) δ 12.1 (s, 1H), 8.07 (s, 1H),8.03 (d, 1H, J=8.5 Hz), 7.74 (d, 1H, J=7.5 Hz), 7.55 (s, 1H), 7.52 (t,1H, J=7.5 Hz), 7.35 (d, 1H, J=8.5 Hz), 7.30 (d, 1H, J=7.5 Hz), 7.22 (t,1H, J=7.5 Hz), 6.77 (s, 1H), 5.21 (t, 1H, J=5.5 Hz), 4.60 (d, 2H, J=5.5Hz), 1.35 (s, 9H).

[0247] 5-Formyl-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (5-8)

[0248] The pre-activated MnO₂ (34.5 g, 15 equiv) and the alcohol (5-7,10.32 g, 1.0 equiv) were weighed into a 1 liter flask and suspended indry dichloromethane (500 mL). The reaction mixture was heated to 45 degC. and was complete by thin layer chromatography after 1 h. The mixturewas allowed to cool to ambient temperature and the manganese oxide(s)were removed by vacuum filtration. The resulting pad of oxides on thefilter were triturated with hot THF and the solvent filtered throughunder vacuum to remove any product from the oxides. The resultingfiltrate was concentrated in vacuo to afford the crude aldehyde as ayellow solid. The solid was triturated with methanol (10 mL) and ethylacetate (15 mL) followed by vacuum filtration to isolate the pureproduct. The light-yellow aldehyde was dried under vacuum (5-8, 9.84 g,96%); ¹H NMR (500 MHz, DMSO-d₆) δ 12.15 (s, 1H), 10.08 (s, 1H), 8.26 (d,1H, J=1.5 Hz), 8.24 (d, 1H, J=8.5 Hz), 8.15 (s, 1H), 7.90 (dd, 1H,J=8.5, 1.5 Hz), 7.77 (d, 1H, J=7.5 Hz), 7.55 (m, 1H), 7.37 (d, 1H, J=8.5Hz), 7.24 (m, 1H), 7.01 (s, 1H).

[0249]5-[4-(2-Hydroxy-ethanoyl)-piperazin-1-ylmethyl]-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (5-9)

[0250] To a stirred solution of the aldehyde (5-8, 2.01 g, 5.15 mmol, 1equiv) and N-(2-hydroxyacetyl)piperazine (2.97 g, 20.60 mmol, 4 equiv)in dichloroethane (400 mL) was added at ambient temperature acetic acid(1.2 mL). The reaction mixture was treated with sodiumtriacetoxyborohydride and stirred for 3 h. The reaction stopped at 76%of conversion and treated with MgSO₄ and additional 1 g of the hydride.After further stirring for 1 h the reaction was complete. The reactionmixture was partitioned between ethyl acetate and saturated aqueousNaHCO₃. The organic layer was once again washed with saturated aqueousNaHCO₃, and then with brine, separated, dried with (Na₂SO₄) andconcentrated in vacuo. The crude solid was dissolved inN,N-dimethylformamide and treated with the activated carbon. Thefiltrate solution (celite) was concentrated to syrup which was quicklytriturated with methanol (100 mL). The resulting solid was collected byfiltration, redissolved in N,N-dimethylformamide, concentrated to syrup,triturated with methanol (100 mL), collected by filtration andvacuum-dried to give5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (5-9, 1.51 g, 57%) as a white powder; ¹H NMR (500MHz, DMSO-d₆) δ 12.24 (br s, 1H), 8.21 (d, 1H, J=8.7 Hz), 7.91 (s, 1H),7.61 (d, 1H, J=6.9 Hz), 7.52 (s, 1H), 7.49 (m, 1H), 7.36 (d, 1H, J=8.1Hz), 7.33 (dd, 1H, J=8.4, 1.5 Hz), 7.24 (m, 1H), 6.67 (s, 1H), 4.15 (s,2H), 3.69 (m, 2H), 3.65 (s, 2H), 3.28 (m, 2H), 2.49 (m, 4H), 1.40 (s,9H).

[0251]3-{5-[4-(2-Hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one(5-10)

[0252] A mixture of5-(4-Methanesulfonyl-piperazin-1-ylmethyl)-2-(2-oxo-1,2-dihydro-quinolin-3-yl)-indole-1-carboxylicacid tert-butyl ester (5-9, 1.05 g, 2.033 mmol), dimethylsulfide (1.2mL), water (0.6 mL) and trifluoroacetic acid (40 mL) in dichloromethane(40 mL) was stirred for 1.5 h. The reaction mixture was concentrated invacuo, partitioned between ethyl acetate and saturated aqueous NaHCO₃.The organic layer was washed with brine, separated, dried (Na₂SO₄), andconcentrated in vacuo. The resulting crude solid was purified byreverse-phase liquid chromatography (H₂O/CH₃CN gradient with 0.1% TFApresent) to give trifluoroacetic acid salt of 5-10. All the fractionscontaining the desired product was partitioned between ethyl acetate andsaturated aqueous NaHCO₃ The organic layer was washed with brine,separated, dried (Na₂SO₄), and concentrated in vacuo to give3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one(5-10, 737 mg, 87%) as a bright yellow solid; ¹H NMR (500 MHz, DMSO-d₆)δ 12.16 (br s, 1H), 11.53 (s, 1H), 8.52 (s, 1H), 7.73 (d, 1H, J=7.5 Hz),7.52 (dt, 1H, J=8.5, 1.0 Hz), 7.47 (d, 1H, J=9.0 Hz), 7.46 (s, 1H), 7.38(d, 1H, J=8.0 Hz), 7.29 (d, 1H, J=1.0 Hz), 7.25 (t, 1H, J=7.5 Hz), 7.08(dd, 1H, J=8.0, 1.0 Hz), 4.51 (t, 1H, J=5.5 Hz), 4.06 (d, 1H, J=5.5 Hz)3.55 (s, 2H), 3.46 (m, 2H), 3.32 (m, 2H), 2.36 (m, 4H).

[0253]4-(2-Hydroxy-ethanoyl)-1-[2-(2-oxo-1,2-dihydro-quinolin-3-yl)-1H-indol-5-ylmethyl]-piperazin-1-ium;methanesulfonate (3-10B)

[0254] To a stirred suspension of the free base (3-10, 9.22 mmol) inMeOH (2 L) was slowly added at rt a 0.3M-MsOH (30.73 mL, 1.0 equiv.,9.22 mmol). After all the solid dissolved, the mixture was filtered intoa rb flask and concentrated on vacuo (with the bath temp ˜10° C.). Theresulting solid was suspended with 200 mL of ethyl acetate, filtered,dried to afford the desired Ms salt(3-10B); ¹H NMR (500 MHz, DMSO-d₆) δ12.21 (s, 1H), 11.81 (s, 1H), 9.70 (br s, 1H), 8.59 (s, 1H), 7.75 (d,1H, J=8.0 Hz), 7.72 (s, 1H), 7.62 (d, 1H, J=8.5 Hz), 7.54 (t, 1H,J=7.5), 7.39 (d, 1H, J=8.0 Hz), 7.39 (s, 1H), 7.27 (t, 1H, J=7.5 Hz),7.22 (d, 1H, J=8.5 Hz), 4.83 (br s, 1H), 4.42 (br s, 3H), 4.17 (d, 1H,J=15.0 Hz), 4.07 (d, 1H, J=15.0 Hz), 3.94 (d, 1H, J=13.5 Hz), 3.34 (s,3H), 3.10 (m, 1H), 2.97 (m, 2H), 2.30 (m, 3H).

[0255] 4-(2-Hydroxy-ethanoyl)1-[2-(2-oxo-1,2-dihydro-quinolin-3-yl)-1H-indol-5-ylmethyl]-piperazin-1H-ium;chloride (3-10A)

[0256] To a stirred suspension of the free base (3-10, 0.465 mmol) inMeOH (200 mL) was slowly added at rt a 1N-HCl (0.47 mL, 1.0 equiv.,0.465 mmol). After all the solid dissolved, the mixture was filteredinto a rb flask and concentrated on vacuo (with the bath temp ˜10° C.).The resulting solid was suspended with 200 mL of ethyl acetate,filtered, dried to afford the desired HCl salt (3-10A); ¹H NMR (500 MHz,DMSO-d₆) δ 12.21 (s, 1H), 11.82 (s, 1H), 10.59 (br s, 1H), 8.60 (s, 1H),7.75 (s, 1H), 7.74 (d, 1H, J=8.5 Hz), 7.61 (d, 1H, J=8.5 Hz), 7.54 (t,1H, J=8.5), 7.40 (d, 1H, J=8.5 Hz), 7.39 (s, 1H), 7.29 (m, 1H), 7.26 (t,1H, J=7.0 Hz), 4.40 (m, 3H), 4.16 (d, 1H, J=14.5 Hz), 4.06 (d, 1H,J=14.5 Hz), 3.92 (d, 1H, J=13.0 Hz), 3.42 (m, 2H), 3.17 (s, 1H), 3.06(m, 2H), 2.96 (m, 1H).

[0257] Characteristics of the Free Base

[0258] The free base is a yellow powder which contains particles thatare birefringent under polarized light indicating the presence ofcrystalline material. TGA analysis with a scan rate of 10° C./min to350° C. shows the solid loses 0.67% weight up to 125° C. and decomposesabove 300° C. DSC analysis with a scan rate of 10° C./min to 350° C.shows a reversible endotherm at 299° C. indicating that the soliddecomposes upon melting. The aqueous solubility was measured using HPLCto quantitate the compound in an aqueous suspension. The aqueoussolubility at room temperature is 0.0635 mg/mL.

[0259] HCl Salt

[0260] The HCl salt is also a yellow solid, which contains particlesthat are birefringent under polarized light indicating the presence ofcrystalline material. TGA analysis with a scan rate of 10° C./min to350° C. shows the solid loses 4.92% weight up to 125° C. and decomposesabove 300° C. DSC analysis with a scan rate of 10° C./min to 350° C.shows a reversible endotherm at 235° C. The aqueous solubility wasmeasured using HPLC to quantitate the compound in suspension. Theaqueous solubility at room temperature is 1.31 mg/mL.

[0261] Mesylate Salt

[0262] The yellow, mesylate salt was examined under polarized light byoptical microscopy. The solid does not show birefringent particles underpolarized light indicating the presence of amorphous material. TGAanalysis with a scan rate of 10° C./min to 350° C. shows the solid loses4.89% weight up to 125° C. and decomposes above 300° C. DSC analysiswith a scan rate of 10° C./min to 350° C. shows a reversible endothermat 153° C. It is possible that the small amount of crystalline solidwent undetected by optical microscopy. The aqueous solubility wasmeasured using HPLC to quantitate the compound in suspension. Theaqueous solubility at room temperature is 2.43 mg/mL.

EXAMPLE 4

[0263] Salts of3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)-2(1H)-quinolinone4-9

[0264] 2-chloro-3-iodo-quinoline (1-2)

[0265] A suspension of 3-(2-chloro)-quinolineboronic acid (1-1, 5.05 g,24.3 mmol, 1 equiv, prepared by the method of Marsais, F; Godard, A.;Queguiner, G. J. Heterocyclic Chem. 1989, 26, 1589-1594) andN-iodosuccinimide (5.48 g, 24.4 mmol, 1.00 equiv) in acetonitrile (300mL) was stirred at 23° C. in the dark for 20 hours. The reaction mixturewas concentrated to dryness, and the resulting yellow solid waspartitioned between saturated aqueous sodium bicarbonate solution anddichloromethane. The organic layer was washed with water, then driedover magnesium sulfate and concentrated to give2-chloro-3-iodo-quinoline as a pale yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.67 (s, 1H), 7.99 (br d, 1H, J=8.4 Hz), 7.75 (br t, 1H, J=7.7Hz), 7.72 (br d, 1H, J=7.8 Hz), 7.57 (br t, 1H, J=7.6 Hz).

[0266] 5-(tert-butyl-dimethyl-silanyloxy-1H-indole (1-4)

[0267] A solution of 5-hydroxyindole (1-3, 5.50 g, 41.3 mmol, 1 equiv),tert-butyldimethylsilyl chloride (7.47 g, 49.6 mmol, 1.20 equiv), andimidazole (7.03 g, 103 mmol, 2.50 equiv) in N, N-dimethylformamide (20mL) was stirred at 23° C. for 20 hours. The reaction mixture wasconcentrated, and the residue was partitioned between ethyl acetate andwater. The organic layer was washed with water (3×), then dried overmagnesium sulfate and concentrated. The residue was purified by flashcolumn chromatography (40% dichloromethane in hexanes, then 60%dichloromethane in hexanes) to give5-(tert-butyl-dimethyl-silanyloxy)-1H-indole as a colorless oil whichsolidified upon standing. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (br s, 1H),7.22 (d, 1H, J=8.7 Hz), 7.17 (t, 1H, J=2.8 Hz), 7.06 (d, 1H, J=2.3 Hz),6.76 (dd, 1H, J=8.6, 2.3 Hz), 6.44 (m, 1H), 1.00 (s, 9H), 0.19 (s, 6H).

[0268] 5-(tert-butyl-dimethyl-silanyloxy)-indole-1-carboxylic acidtert-butyl ester (1-5)

[0269] A solution of 5-(tert-butyl-dimethyl-silanyloxy)-1H-indole (1-4,10.2 g, 41.3 mmol, 1 equiv), di-tert-butyl dicarbonate (14.4 g, 66.0equiv, 1.60 equiv), and 4-dimethylaminopyridine (1.01 g, 8.25 mmol,0.200 equiv) in dichloromethane (100 mL) was stirred at 23° C. for 20hours. The reaction mixture was concentrated, and the residue waspurified by flash column chromatography (40% dichloromethane in hexanes)to afford 5-(tert-butyl-dimethyl-silanyloxy)-indole-1-carboxylic acidtert-butyl ester (1-5) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ7.96 (br d, 1H, J=7.5 Hz), 7.54 (br d, 1H, J=3.1 Hz), 6.98 (d, 1H, J=2.4Hz), 6.83 (dd, 1H, J=9.0, 2.4 Hz), 6.45 (d, 1H, J=3.7 Hz), 1.66 (s, 9H),1.00 (s, 9H), 0.20 (s, 6H).

[0270]1-(tert-butoxycarbonyl)-5-{[tert-butyl(dimethyl)silyl]oxy}-1H-indol-2-ylboronicacid (1-6)

[0271] A solution of tert-butyllithium in pentane (1.7 M, 20.7 mL, 35.2mmol, 1.20 equiv) was added to a solution of5-(tert-butyl-dimethyl-silanyloxy)-indole-1-carboxylic acid tert-butylester (1-5, 10.2 g, 29.3 mmol, 1 equiv) in tetrahydrofuran (100 mL) at−78° C. The resulting light-brown solution was stirred at −78° C. for 30minutes, then trimethylborate (6.67 mL, 58.7 mmol, 2.00 equiv) wasadded. The resulting mixture was warmed to 0° C., then diluted withsaturated aqueous ammonium chloride solution (100 mL) and ethyl ether(200 mL). The aqueous layer was made acidic with aqueous 10% potassiumhydrogensulfate solution. The organic layer was separated, then washedwith brine, dried over magnesium sulfate, and concentrated. The residualyellow solid was triturated with hexanes to give1-(tert-butoxycarbonyl)-5-{[tert-butyl(dimethyl)silyl]oxy}-1H-indol-2-ylboronicacid (1-6) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.84 (d, 1H,J=8.9 Hz), 7.37 (s, 1H), 7.01 (d, 1H, J=2.4 Hz), 6.97 (br s, 2H), 6.88(dd, 1H, J=9.0, 2.4 Hz), 1.73 (s, 9H), 1.00 (s, 9H), 0.20 (s, 6H).

[0272] tert-butyl5-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-chloro-3-quinolinyl)-1H-indole-1-carboxylate(1-7)

[0273] A deoxygenated mixture of 1-(tert-butoxycarbonyl)-5-{[tert-butyl(dimethyl)silyl]oxy}-1H-indol-2-ylboronic acid (1-6, 4.10 g, 10.5 mmol,1 equiv), 2-chloro-3-iodo-quinoline (1-2, 3.64 g, 12.6 mmol, 1.20equiv), potassium phosphate (6.67 g, 31.4 mmol, 3.00 equiv), andtetrakis(triphenylphosphine)palladium (0.605 g, 0.524 mmol, 0.050 equiv)in dioxane (100 mL) was heated at 90° C. for 20 hours. The reactionmixture was cooled, then partitioned between a mixture of water andethyl acetate. The organic layer was separated, washed with brine, driedover magnesium sulfate, and concentrated. The residue was purified byflash column chromatography (20% dichloromethane in hexanes, grading to90% dichloromethane in hexanes) to give tert-butyl5-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-chloro-3-quinolinyl)-1H-indole-1-carboxylate(1-7) as a tan-colored foam. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H),8.15 (d, 1H, J=9.0 Hz), 8.07 (d, 1H, J=8.2 Hz), 7.86 (d, 1H, J=7.8 Hz),7.77 (br t, 1H, J=8.4 Hz), 7.60 (br t, 1H, J=8.1 Hz), 7.03 (d, 1H, J=2.4Hz), 6.92 (dd, 1H, J=9.0, 2.4 Hz), 6.55 (s, 1H), 1.26 (s, 9H), 1.02 (s,9H), 0.23 (s, 6H).

[0274] tert-butyl 2-(2-chloro-3-quinolinyl)-5-hydroxy-1H-indole-l-carboxylate (1-8)

[0275] A solution of tert-butyl 5-{[tert-butyl(dimethyl)silyl]oxy}-2-(2-chloro-3-quinolinyl)-1H-indole-1-carboxylate (1-7, 2.50 g, 4.91mmol, 1 equiv) and triethylamine trihydrofluoride (3.60 mL, 22.1 mmol,4.50 equiv) in acetonitrile (100 mL) was stirred at 23° C. for 20 hours.The reaction mixture was concentrated, and the residue was partitionedbetween saturated aqueous sodium bicarbonate solution and ethyl acetate.The organic layer was washed with brine, dried over magnesium sulfateand concentrated to tert-butyl2-(2-chloro-3-quinolinyl)-5-hydroxy-1H-indole-1-carboxylate (1-8) as atan colored foam (2.1 g, 100%). ¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, 1H,J=9.0 Hz), 8.17 (s, 1H), 8.07 (d, 1H, J=8.4 Hz), 7.86 (d, 1H, J=8.1 Hz),7.77 (br t, 1H, J=8.4 Hz), 7.61 (br t, 1H, J=8.1 Hz), 7.03 (d, 1H, J=2.6Hz), 6.93 (dd, 1H, J=8.8, 2.6 Hz), 6.55 (s, 1H), 1.26 (s, 9H).

[0276]3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one(1-9)

[0277] A mixture of tert-butyl2-(2-chloro-3-quinolinyl)-5-hydroxy-1H-indole-1-carboxylate (1-8, 1.50g, 3.80 mmol, 1 equiv), 2-chloro-N-(2-methoxyethyl)-N-methylethanamine(720 mg, 4.75 mmol, 1.25 equiv), and cesium carbonate (3.09 g, 9.50mmol, 2.50 equiv) in N,N-dimethylformamide (20 mL) was heated at 70° C.for 5 hours. The reaction mixture was concentrated, and the residue waspartitioned between water and ethyl acetate. The organic layer waswashed with water then brine, dried over magnesium sulfate, andconcentrated to give a brown gum. The gum was dissolved in a 1:1 mixtureof water and acetic acid (60 mL), and the resulting solution was heatedat 100° C. for 18 hours. The reaction mixture was concentrated, and theresidue was partitioned between aqueous saturated sodium bicarbonatesolution and ethyl acetate. The organic layer was washed with water thenbrine, dried over magnesium sulfate, and concentrated to give a yellowsolid. Purification by flash column chromatography (5% ethanol saturatedwith ammonia/CH₂Cl₂, grading to 10% ethanol saturated withammonia/CH₂Cl₂) provided 3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one (1-9) as a yellowsolid. ¹H NMR (400 MHz, CDCl₃) δ 11.10 (s, 1H), 9.72 (s, 1H), 8.32 (s,1H), 7.68 (br d, 1H, J=7.8 Hz), 7.53 (br t, 1H, J=7.6 Hz), 7.35 (d, 1H,J=8.8 Hz), 7.29 (br t, 1H, J=7.8 Hz), 7.24 (br d, 1H, J=8.2 Hz), 7.09(d, 1H, J=2.2 Hz), 6.97 (d, 1H, J=1.4 Hz), 6.89 (dd, 1H, J=8.6, 2.2 Hz),4.16 (t, 2H, J=5.9 Hz), 3.54 (t, 2H, J=2.67 (t, 3H, J=5.7 Hz), 3.38 (s,3H), 2.92 (t, 3H, J=6.0 Hz), 2.73 (t, 3H, J=5.8 Hz), 2.44 (s, 3H).

[0278]2-methoxy-N-methyl-N-(2-{[2-(2-oxo-1,2-dihydroquinolin-3-yl)-1H-indol-5-yl]oxy}ethyl)ethanaminiummethanesulfonate (4-9A)

[0279] Methanesulfonic acid (0.250 mL, 3.83 mmol, 1.00 equiv) was addedto a solution of3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one (4-9, 1.50 g, 3.83 mmol, 1 equiv) in dichloromethane(100 mL) at 23° C. The mixture was concentrated and the residue wassuspended in ethyl ether, filtered, and dried to give2-methoxy-N-methyl-N-(2-{[2-(2-oxo-1,2-dihydroquinolin-3-yl)-1H-indol-5-yl]oxy}ethyl)ethanaminiummethanesulfonate (1-10) as a yellow solid. ¹H NMR (400 MHz, (CD₃)₂SO) δ12.17 (s, 1H), 11.53 (s, 1H), 9.55 (br s, 1H), 8.53 (s, 1H), 7.73 (br d,1H, J=7.9 Hz), 7.52 (br t, 1H, J=7.6 Hz), 7.47 (d, 1H, J=8.6 Hz), 7.38(br d, 1H, J=8.2 Hz), 7.25 (br t, 1H, J=7.7 Hz), 7.25 (br s, 1H), 7.15(d, 1H, J=2.2 Hz), 6.84 (dd, 1H, J=8.6, 2.2 Hz), 4.35 (t, 2H, J=4.9 Hz),3.71 (t, 3H, J=4.9 Hz), 3.64 (m, 1H), 3.52 (m, 3H), 3.33 (s, 3H), 2.92(br s, 3H), 2.30 (s, 3H).

[0280] This mesylate salt 4-9A, which is a yellow powder, was examinedunder polarized light by optical microscopy. The crystals appearbirefringent under polarized light indicating crystallinity. Thecrystals appear plate-like with few agglomerates. TGA analysis at a scanrate of 10° C./min to 350° C. shows the solid loses 0.91% weight up to125° C. and decomposes above 275° C. DSC analysis with a scan rate of10° C./min to 350° C. shows multiple reversible endotherms (82° C.,151.4° C. and 229° C.). The aqueous solubility was measured using HPLCto quantitate the compound in suspension. The solubility of Salt 4-9A atroom temperature is 1.5 mg/mL.

[0281] Two forms of the mesylate salt were made: Salt 4-9A above andSalt 4-9B below. Numerous salts of 4-9 were prepared in situ andanalyzed using the procedure outlined below,

[0282] 1) 1.25×10⁻⁵ moles of free base were placed in a centrifuge tube.

[0283] 2) The free base was then reacted with 1.05 mole equivalents ofacid.

[0284] 3) The reagents were mixed with a Vortex mixer and left to standat room temperature or warmed if needed to dissolve the solid.

[0285] 4) 100 μL of water were added to suspend the solid.

[0286] 5) Tube was then covered with aluminum foil and spun overnight ona rotator.

[0287] 6) The tube was then spun in a centrifuge for 10 minutes at10,000 RPM.

[0288] 7) An aliquot was removed from the sample and dried with nitrogenovernight to produce the solid residue (salt) which was analyzed viamicroscopy and DCS.

[0289] 8) The liquid residue from the remaining sample was used tomeasure pH and determine the concentration of salt by HPLC.

[0290] The solubility properties of these salts are summarized in TableVII below. TABLE VII Solubility of Salts of 4-9 Salt Solubility (mg/mL)pH Free Base 0.075 6.75 Mesylate 4-9B 22.0 5.08 HCl 22.4 2.34 Tartrate25.3 3.23 Citrate 19.8 3.33 Sulfate 4.35 1.40

What is claimed is:
 1. A mesylate salt3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.2. A chloride salt3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.3. The mesylate salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-oneaccording to claim 1 in crystalline form characterized by an X-raypowder diffraction pattern having diffraction angles of: 7.39, 8.20,9.03, 9.90, 10.94, 15.45, 17.12, 17.84, 18.29, 18.64, 19.24, 19.77,20.28, 21.73, 22.49, 23.27, 24.15, 24.73, 25.40, 26.79, and 27.50. 4.The mesylate salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-oneaccording to claim 1 in crystalline form characterized by an X-raypowder diffraction pattern having diffraction angles of: 6.94, 8.01,9.74, 10.47, 10.77, 11.75, 12.61, 14.02, 15.28, 15.86, 16.93, 17.61,18.69, 19.04, 19.47, 20.11, 21.56, 21.94, 22.53, 23.85, and 27.22. 5.The chloride salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-oneaccording to claim 2 in crystalline form characterized by an X-raypowder diffraction pattern having diffraction angles of: 7.08, 7.86,8.99, 14.54, 15.40, 16.14, 16.81, 18.06, 19.91, 20.72, 22.72, 24.11,26.09, 28.67, and 29.89.
 6. The chloride salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-oneaccording to claim 2 in crystalline form characterized by an X-raypowder diffraction pattern having multiple diffraction peaks between 5°and 30° 2-theta and a melting endotherm of 284.08° C. at a rate of 10°C. per minute.
 7. A mesylate salt of3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.8. A chloride salt of 3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one.
 9. A mesylate salt of3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one.10. A chloride salt of3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one.11. The chloride salt of3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-oneaccording to claim 10 in crystalline characterized by a reversibleendotherm at 235° C. at a scan rate of 10° C. per minute.
 12. A mesylatesalt of 3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one.
 13. The mesylate salt of3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one in crystalline formaccording to claim 12 characterized by multiple reversible endotherms at82° C., 151.4° C., and 229° C. at a scan rate of 10° C. per minute. 14.A mesylate or chloride salt of3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one;3-[5-(4-methyl-5-oxo-[1,4]diazepan-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one;3-{5-[4-(2-hydroxy-ethanoyl)-piperazin-1-ylmethyl]-1H-indol-2-yl}-1H-quinolin-2-one;or3-(5-{2-[(2-methoxyethyl)(methyl)amino]ethoxy}-1H-indol-2-yl)quinolin-2(1H)-one.15. A pharmaceutical composition which is comprised of a salt inaccordance with claim 14 and a pharmaceutically acceptable carrier. 16.A method of treating or preventing cancer in a mammal in need of suchtreatment which is comprised of administering to said mammal atherapeutically effective amount of a salt of claim
 14. 17. A method oftreating cancer or preventing cancer in accordance with claim 16 whereinthe cancer is selected from cancers of the brain, genitourinary tract,lymphatic system, stomach, larynx and lung.
 18. A method of treating orpreventing cancer in accordance with claim 16 wherein the cancer isselected from histiocytic lymphoma, lung adenocarcinoma, small cell lungcancers, pancreatic cancer, gioblastomas and breast carcinoma.
 19. Amethod of treating or preventing a disease in which angiogenesis isimplicated, which is comprised of administering to a mammal in need ofsuch treatment a therapeutically effective amount of a salt of claim 14.20. A method in accordance with claim 19 wherein the disease is anocular disease.
 21. A method of treating or preventing retinalvascularization which is comprised of administering to a mammal in needof such treatment a therapeutically effective amount of a salt of claim14.
 22. A method of treating or preventing diabetic retinopathy which iscomprised of administering to a mammal in need of such treatment atherapeutically effective amount of a salt of claim
 14. 23. A method oftreating or preventing age-related macular degeneration which iscomprised of administering to a mammal in need of such treatment atherapeutically effective amount of a salt of claim
 14. 24. A method oftreating or preventing inflammatory diseases which comprisesadministering to a mammal in need of such treatment a therapeuticallyeffective amount of a salt of claim
 14. 25. A method according to claim24 wherein the inflammatory disease is selected from rheumatoidarthritis, psoriasis, contact dermatitis and delayed hypersensitivityreactions.
 26. A method of treating or preventing a tyrosinekinase-dependent disease or condition which comprises administering atherapeutically effective amount of a salt of claim
 14. 27. Apharmaceutical composition made by combining the salt of claim 14 and apharmaceutically acceptable carrier.
 28. A process for making apharmaceutical composition which comprises combining a salt of claim 14with a pharmaceutically acceptable carrier.
 29. A method of treating orpreventing bone associated pathologies selected from osteosarcoma,osteoarthritis, and rickets which comprises administering atherapeutically effective amount of a salt of claim
 14. 30. Thecomposition of claim 15 further comprising a second compound selectedfrom: 1) an estrogen receptor modulator, 2) an androgen receptormodulator, 3) retinoid receptor modulator, 4) a cytotoxic agent, 5) anantiproliferative agent, 6) a prenyl-protein transferase inhibitor, 7)an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor, 9) areverse transcriptase inhibitor, and 10) another angiogenesis inhibitor.31. The composition of claim 30, wherein the second compound is anotherangiogenesis inhibitor selected from the group consisting of a tyrosinekinase inhibitor, an inhibitor of epidermal-derived growth factor, aninhibitor of fibroblast-derived growth factor, an inhibitor of plateletderived growth factor, an MMP inhibitor, an integrin blocker,interferon-α, interleukin-12, pentosan polysulfate, a cyclooxygenaseinhibitor, carboxyamidotriazole, combretastatin A-4, squalamine,6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,troponin-1, and an antibody to VEGF.
 32. The composition of claim 30,wherein the second compound is an estrogen receptor modulator selectedfrom tamoxifen and raloxifene.
 33. A method of treating cancer whichcomprises administering a therapeutically effective amount of a salt ofclaim 14 in combination with radiation therapy.
 34. A method of treatingor preventing cancer which comprises administering a therapeuticallyeffective amount of a salt of claim 14 in combination with a compoundselected from: 1) an estrogen receptor modulator, 2) an androgenreceptor modulator, 3) retinoid receptor modulator, 4) a cytotoxicagent, 5) an antiproliferative agent, 6) a prenyl-protein transferaseinhibitor, 7) an HMG-CoA reductase inhibitor, 8) an HIV proteaseinhibitor, 9) a reverse transcriptase inhibitor, and 10) anotherangiogenesis inhibitor.
 35. A method of treating cancer which comprisesadministering a therapeutically effective amount of a salt of claim 14in combination with radiation therapy and a compound selected from: 1)an estrogen receptor modulator, 2) an androgen receptor modulator, 3)retinoid receptor modulator, 4) a cytotoxic agent, 5) anantiproliferative agent, 6) a prenyl-protein transferase inhibitor, 7)an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor, 9) areverse transcriptase inhibitor, and 10) another angiogenesis inhibitor.36. A method of treating or preventing cancer which comprisesadministering a therapeutically effective amount of a salt of claim 14and paclitaxel or trastuzumab.
 37. A method of treating or preventingcancer which comprises administering a therapeutically effective amountof a salt of claim 14 and a GPIIb/IIIa antagonist.
 38. The method ofclaim 37 wherein the GPIIb/IIIa antagonist is tirofiban.
 39. A method ofreducing or preventing tissue damage following a cerebral ischemic eventwhich comprises administering a therapeutically effective amount of asalt of claim 14.